Physiologically active protein originating in mammals

ABSTRACT

The present invention provides novel physiologically active protein molecules originating in mammals, which are specifically expressed in arteriosclerosis and/or coronary restenosis, and are predicted to relate closely to the onset and progress of these diseases; DNAS encoding the protein molecules; antibodies reactive with the molecules; and pharmaceutical compositions comprising the above protein molecule or the antibody. The protein molecules, DNAs, and antibodies are useful for treating and preventing arteriosclerosis.

TECHNICAL FIELD

[0001] The present invention relates to a novel physiologically activeprotein originating in mammals, a DNA encoding said protein, and anantibody reactive with said protein.

BACKGROUND ART

[0002] A so-called geriatric disease, which is regarded as a currentdisease in high living standard society, includes arteriosclerosis aswell as hypertension and diabetes. Important measures for preventingthese diseases are not only development of therapeutic methods but alsodaily life control.

[0003] Arteriosclerosis begins with pathological changes (for example,(1) invasive growth of smooth muscle cells into inner membrane, (2)qualitative and quantitative changes of collagen, elastin, and acidicmucopolysaccharides, and (3) cell foaming by lipid accumulation in thecytoplasm of grown smooth muscle cells and macrophages implantingtissues) occurring in inner membrane of artery. As the result of suchpathological changes, (1) foam cells found in the inner membraneproduces fat spots on the surface of the inner membrane, (2) lipidaccumulates between tissues (deep part of midmembrane) and the innermembrane surface is covered with thick glass-like membrane, accompaniedby fibrous growth and calcification, and (3) bleeding and necrosis occurin tissues to cause combined pathological changes involvingthrombogenesis, calcification, and deposition of lipid crystals. Suchpathological changes, in time, distribute in artery of a whole body andnarrow the cavity of the artery. In addition, the site of pathologicalchanges becomes bursal and the vascular wall loses elasticity, therebyhardening blood vessels. The vessels then wind, and normal blood flow isinhibited.

[0004] Epidemiological studies so far have illustrated age (aboutthirties or more), hypercholesterolemia, hypo-HDL-cholesterolemia,systolic hypertension, obesity, hemoglobin high value, and diabetes asrisk factors of the onset of arteriosclerosis. Dynamics of in vivofactors inducing the onset include secretion of adrenalin, increase ofthromboxane A2, decrease of prostacyclin, increase of serum peroxylipid,increase of free fatty acid, increase of platelet, increase offibrinogen, increase of blood coagulation factors (XII and XIII),decrease of tissue plasmin, increase of prostaglandin, decrease ofantithrombin III, increase of serum LDL, decrease of serum HDL, increaseof insulin, and increase of renin.

[0005] Studies so far have revealed only that multiple conditions, forexample, physical conditions such as age and obesity, complication withother diseases, and abnormalities of the dynamics of many in vivofactors complicatedly are related to each other to causearteriosclerosis.

[0006] Treatments of arteriosclerosis are divided with their purposeinto (1) preventive treatments to retract arteriosclerosis and toprevent the onset of arteriosclerosis by correcting lifestyle andphysical abnormalities such as obesity (for example, diet therapy andtherapeutic exercise) and (2) chemotherapy or surgical therapy to removevessel occlusion symptoms occurring with the progress ofarteriosclerosis or to prevent the onset of vessel cavity occlusionsymptoms by thrombus or embolus,.

[0007] Since particular decisive causes of arteriosclerosis are unclear,only symptomatic treatment by chemotherapy is currently possible. Forexample, β blocker is applied when the enhancement of a catecholaminederivative such as adrenalin is suspected as the cause, eicosapentaenoicacid is applied for a prostaglandin derivative, vitamin E is applied forperoxylipid, and urokinase is applied for thrombus. No effectivepharmaceuticals for treating the arteriosclerosis have been providedyet.

[0008] In the surgical therapy for arterial occulsion, percutaneoustransluminal coronary angioplasty (PTCA) based on the observation byangiography prevails clinically as an effective means to enlarge vesselcavity. PTCA has remarkably progressed and prevailed since it wasclinically applied by Gruntzig for the first time in 1977, and thenumber of the operation has rapidly increased in Japan.

[0009] PTCA is the method in which the occlusion (constriction) site isenlarged by inserting a thin catheter with a balloon at the tip in athick catheter into the coronary artery occlusion site and by expandingthe balloon.

[0010] However, in cavity enlargement by PTCA, restenosis occurs at theoperation site of the artery in about 30 to 50% of the cases within afew months after the operation, and this restenosis is a major drawbackof PTCA.

[0011] The restenosis has been thought to occur by the amplification ofneonatal inner membrane proliferation based on the repair reaction ofthe injury site of the vascular wall, which has been inevitably causedby the enlargement of the occlusion site by PTCA. Although chemotherapyhas been tried for preventing this restenosis, almost no effective drugshave been reported so far.

[0012] As mentioned above, at present, a method for the completetreatment and prevention of arteriosclerosis comprising the preventionof the recurrence of arteriosclerosis and the occurrence of restenosishas not established. It is thus desired to clarify the cause of theonset and progress of arteriosclerosis and to develop a method for theeffective treatment and prevention thereof, and therapeutic andpreventive drugs.

[0013] Coronary artery restenosis occurring after PTCA is regarded as aclinical model of arteriosclerosis from pathological viewpoints such asneonatal inner membrane proliferation or intimal thickening. Therefore,to diagnose the tissue characteristics of the vascular wall at therestenosis site after PTCA and to elucidate the difference between thecharacteristics and those of normal vascular wall by comparing thempathologically and at the gene level are effective to identify the causeand factors of restenosis, and further, arteriosclerosis.

[0014] In such comparative studies, a useful method for comparison andexamination at the gene level using the genetic engineering technique iscalled differential display method (Nucleic Acids Research, Vol.21,No.18, pp.4272-4280 (1993); and Science, Vol.257, pp.967-971 (1992)).

[0015] Specifically, PCTA is applied to the coronary artery of a largemammal such as a rabbit, the expression patterns of genes in the innermembrane tissue at the PTCA site are examined by differential displaymethod, and they are compared with the gene expression patterns in theinner membrane tissue without PCTA, to thereby identify genesspecifically or increasingly expressed after PTCA.

DISCLOSURE OF THE INVENTION

[0016] Genes that express specifically or increasingly after PTCA andproteins derived from said genes may be closely related toarteriosclerosis and restenosis. The present invention providespharmaceuticals and methods for preventing and treating arteriosclerosisand restenosis by identifying genes and proteins expressing specificallyin arteriosclerosis and coronary artery restenosis.

[0017] As the result of studies on the analyses of genes specific toarteriosclerosis and/or coronary artery restenosis, the presentinventors have discovered genes encoding two novel proteins (cloneBA0306 and BA2303) that express increasingly at the comparatively earlystage (day 1 to 7) after PTCA and completed the present invention.

[0018] The two novel protein-encoding genes of the present invention,whose characteristics are mentioned below, are expressed specificallyafter PTCA, and are thought to be genes involved in onset and progressof arteriosclerosis and/or coronary artery restenosis.

[0019] Clone BA0306 has the following characteristics.

[0020] (1) Its increased expression is observed on day 1 to 7 after PTCAof coronary artery (the peak is observed on day 4).

[0021] (2) Northern blotting reveals the expression of the mRNA as about3.5 k and about 4.4 k bands in various human tissues.

[0022] (3) It has ten putative transmembrane regions.

[0023] (4) It has amino acid sequence homology with S. cerevisiaeoxidative stress resistance protein, S. cerevisiae zinc/cadmiumresistance protein, heavy metal ion resistance protein, and so on.

[0024] (5) The molecules derived from humans and rabbits have the aminoacid sequences of SEQ ID NO: 10 and 8, respectively. The moleculederived from mice has the amino acid sequence of SEQ ID NO: 28.

[0025] Judging from these characteristics, clone BA0306 is thought toinhibit active oxygen such as nitrogen monoxide (NO), which is involvedin the progress of arteriosclerosis and/or restenosis.

[0026] Clone BA2303 has the following characteristics.

[0027] (1) Its increased expression is observed from day 1 after PTCA ofcoronary artery, and the expression continues until day 7 with themaximum expression on day 2 to 4.

[0028] (2) Northern blotting reveals the expression of the mRNA as about3.9 k and about 2.1 k bands in various human tissues.

[0029] (3) It has seven putative transmembrane regions.

[0030] (4) The molecules derived from humans and mice have the aminoacid sequences of SEQ ID NO: 4 and 6, respectively. The molecule derivedfrom rabbits has the amino acid sequence of SEQ ID NO: 2.

[0031] Judging from these characteristics, clone BA2303 is thought to bea GTP binding protein (G protein)-coupled receptor that transmits aspecific signal through intracellular G protein to an effector on theplasma membrane or the surface of the cytoplasm by binding to an in vivoligand involved in the onset or progress of arteriosclerosis and/orrestenosis.

[0032] Therefore, the genes (DNAs), proteins, or their fragments of thepresent invention and antibodies or a portion of them reactive with theproteins of the present invention are extremely useful for developingdrugs for treatment and prevention of arteriosclerosis and for treatmentand prevention of restenosis after PTCA for artery occlusion symptom andso on, targeting said genes or protein molecules. In addition, the DNAsof the present invention themselves are useful as antisensepharmaceuticals, extracellular region fragments of said proteins, forexample, as soluble receptor pharmaceuticals, and said antibodies and aportion of them as antibody pharmaceuticals.

[0033] Genes (DNAs), proteins, and antibodies of the present inventionare useful as reagents for searching proteins (ligands) interacting withthe proteins of the present invention, thereby elucidating the functionof said ligands, and developing therapeutic drugs targeting saidligands.

[0034] Based on the genetic information of the rabbit- or mouse-derivedDNA, one embodiment of DNAs of the present invention, model animals(knockout animals) can be produced by disrupting (inactivating) theendogenous gene corresponding to the DNA. Similarly, transgenic animalscan be produced as model animals by introducing the human-derived DNA,one embodiment of DNAs of the present invention, into nonhuman mammalssuch as mice. Function of genes and proteins of the present inventioncan be elucidated by analyzing the physical, biological, pathologic, andgenetic characteristics of these model animals.

[0035] Moreover, by mating the model animals whose endogenous gene isthus disrupted with the transgenic animals, model animals that have onlythe human-derived gene of the present invention can be produced. Byadministering drugs (compounds, antibodies, and so on) targeting theintroduced human gene to these model animals, the therapeutic effect ofthe drug can be estimated.

[0036] Namely, the present invention provides the DNAS, proteins,expression vectors, transformants, antibodies, pharmaceuticalcompositions, transgenic mice, and knockout, mentioned below.

[0037] (1) A DNA encoding a protein having the amino acid sequence ofSEQ ID NO: 4.

[0038] (2) A DNA encoding a protein fragment comprising theextracellular region of a protein having the amino acid sequence of SEQID NO: 4.

[0039] (3) A DNA comprising a nucleotide sequence corresponding tonucleotide residues 97 to 1419 of the nucleotide sequence of SEQ ID NO:3.

[0040] (4) A DNA hybridizing with a DNA having the nucleotide sequenceof SEQ ID NO: 3 under stringent conditions.

[0041] (5) A protein having the amino acid sequence of SEQ ID NO: 4 oran amino acid sequence substantially the same as said amino acidsequence.

[0042] (6) A protein fragment comprising the extracellular region of aprotein having the amino acid sequence of SEQ ID NO: 4 or an amino acidsequence substantially the same as said amino acid sequence.

[0043] (7) A fusion protein between the extracellular region of theprotein of (5) and the constant region of the heavy chain of humanimmunoglobulin (Ig) or a portion of the constant region. (8) Anexpression vector comprising the DNA of any one of (1) to (4).

[0044] (9) A transformant carrying the expression vector of (8).

[0045] (10) An antibody or its portion reactive with the protein of (5)or the protein fragment of (6).

[0046] (11) The antibody or its portion of (10), wherein the antibody isa monoclonal antibody.

[0047] (12) A pharmaceutical composition comprising the protein fragmentof (6) or the fusion protein of (7) and a pharmaceutically acceptablecarrier.

[0048] (13) A pharmaceutical composition comprising the antibody or itsportion of (10) or (11) and a pharmaceutically acceptable carrier.

[0049] (14) A DNA encoding a protein having the amino acid sequence ofSEQ ID NO: 10.

[0050] (15) A DNA encoding a protein fragment comprising theextracellular region of a protein having the amino acid sequence of SEQID NO: 10.

[0051] (16) A DNA having a nucleotide sequence corresponding tonucleotide residues 1 to 1785 of the nucleotide sequence of SEQ ID NO:9.

[0052] (17) A DNA hybridizing with a DNA having the nucleotide sequenceof SEQ ID NO: 9 under stringent conditions.

[0053] (18) A protein having the amino acid sequence of SEQ ID NO: 10 oran amino acid sequence substantially the same as said amino acidsequence.

[0054] (19) A protein fragment comprising the extracellular region of aprotein having the amino acid sequence of SEQ ID NO: 10 or an amino acidsequence substantially the same as said amino acid sequence.

[0055] (20) A fusion protein comprising the extracellular region of theprotein of (18) and the constant region of the heavy chain of humanimmunoglobulin (Ig) or a portion of the constant region.

[0056] (21) An expression vector comprising the DNA of any one of (14)to (17).

[0057] (22) A transformant carrying the expression vector of (21).

[0058] (23) An antibody or its portion reactive with the protein of (18)or the protein fragment of (19).

[0059] (24) The antibody or its portion of (23), wherein the antibody isa monoclonal antibody.

[0060] (25) A pharmaceutical composition comprising the protein fragmentof (19) or the fusion protein of (20) and a pharmaceutically acceptablecarrier.

[0061] (26) A pharmaceutical composition comprising the antibody or itsportion of (23) or (24) and a pharmaceutically acceptable carrier.

[0062] (27) A transgenic mouse in which the human-derived DNA comprisinga DNA having a nucleotide sequence corresponding to nucleotide residues97 to 1419 of the nucleotide sequence of SEQ ID NO: 3 is integrated intoan endogenous gene of said mouse.

[0063] (28) A transgenic mouse in which the human-derived DNA comprisinga DNA having a nucleotide sequence corresponding to nucleotide residues1 to 1785 of the nucleotide sequence of SEQ ID NO: 9 is integrated intoan endogenous gene of said mouse.

[0064] (29) A knockout mouse whose endogenous gene encoding amouse-derived protein having the amino acid sequence of SEQ ID NO: 6 isinactivated so that said protein is not produced.

[0065] (30) A knockout mouse whose endogenous gene encoding amouse-derived protein comprising the amino acid sequence of SEQ ID NO:28 is inactivated so that said protein is not produced.

[0066] In the following, the present invention is explained in detail byclarifying the meanings of terms used in the present application and thegeneral production methods of proteins, protein fragments, fusionproteins, DNAs, antibodies, transgenic mice, and knockout mice of thepresent invention.

[0067] A “protein” of the present invention means a protein and itsfragment derived from mammals such as humans, rabbits, and mice, andpreferably, a human-derived protein and its fragment.

[0068] Particularly preferable examples are (1) a protein having theamino acid sequence of SEQ ID NO: 4 or an amino acid sequencesubstantially the same as said amino acid sequence, (2) a proteinfragment comprising the extracellular region of a protein having theamino acid sequence of SEQ ID NO: 4 or an amino acid sequencesubstantially the same as said amino acid sequence, (3) a protein havingthe amino acid -sequence of SEQ ID NO: 10 or an amino acid sequencesubstantially the same as said amino acid sequence, and (4) a proteinfragment comprising the extracellular region of a protein having theamino acid sequence of SEQ ID NO: 10 or an amino acid sequencesubstantially the same as said amino acid sequence.

[0069] The term “extracellular region” used herein is explained below. Atransmembrane protein such as a G protein-coupled receptors or cellsurface molecule connects with the membrane through the hydrophobicpeptide region penetrating the lipid bilayer of the membrane once orseveral times and has structure composed of three main regions, that is,extracellular region, transmembrane region, and cytoplasmic region. Sucha transmembrane protein exists as a monomer, homodimer, heterodimer, oroligomer with another chain(s) having the same or different amino acidsequence.

[0070] The term “extracellular region” used herein means the partialstructure (partial sequence) existing outside of the membrane that holdsthe transmembrane protein as mentioned above among the whole structureof said membrane protein. In other words, it corresponds to the regionexcluding the region incorporated into the membrane (transmembraneregion) and the region existing in the cytoplasm following thetransmembrane region (cytoplasmic region). If desired, one to five aminoacids derived from the amino acids constituting the transmembrane and/orcytoplasmic region can be added to the N-terminus and/or C-terminus ofthe extracellular region in the present invention.

[0071] Here, “having substantially the same amino acid sequence” meansto include a protein having an amino acid sequence where multiple aminoacids, preferably 1 to 10 amino acids, particularly preferably 1 to 5amino acids, in the amino acid sequence shown in SEQ ID NO: 4 or 10, aresubstituted, deleted, and/or modified, and a protein having an aminoacid sequence where multiple amino acids, preferably 1 to 10 aminoacids, particularly preferably 1 to 5 amino acids, are added to saidamino acid sequence, as far as the protein has substantially the samebiological properties as the protein having said amino acid sequence.

[0072] Alphabetical triplet or single letter codes used to representamino acids in the present specification or figures mean amino acids asfollows. (Gly/G) glycine, (Ala/A) alanine, (Val/V) valine, (Leu/L)leucine, (Ile/I) isoleucine, (Ser/S) serine, (Thr/T) threonine, (Asp/D)aspartic acid, (Glu/E) glutamic acid, (Asn/N) asparagine, (Gln/Q)glutamine, (Lys/K) lysine, (Arg/R) arginine, (Cys/C) cysteine, (Met/M)methionine, (Phe/F) phenylalanine, (Tyr/Y) tyrosine, (Trp/W)tryptophane, (His/H) histidine, (Pro/P) proline.

[0073] “The constant region or a portion of the constant region of humanimmunoglobulin (Ig) heavy chain” used herein means the constant regionor the Fc region of human-derived immunoglobulin heavy chain (H chain)as described, or a portion of them. The immunoglobulin can be anyimmunoglobulin belonging to any class and any subclass. Specifically,examples of the immunoglobulin are IgG (IgG1, IgG2, IgG3, and IgG4),IgM, IgA (IgA1 and IgA2), IgD, and IgE. Preferably, the immunoglobulinis IgG (IgG1, IgG2, IgG3, or IgG4), or IgM. Examples of particularlypreferable immunoglobulin of the present invention are those belongingto human-derived IgG (IgG1, IgG2, IgG3, or IgG4).

[0074] Immunoglobulin has a Y-shaped structural unit in which fourchains composed of two homologous light chains (L chains) and twohomologous heavy chains (H chains) are connected through disulfide bonds(S—S bonds). The light chain is composed of the light chain variableregion (VL) and the light chain constant region (CL). The heavy chain iscomposed of the heavy chain variable region (VH) and the heavy chainconstant region (CH).

[0075] The heavy chain constant region is composed of some domainshaving the amino acid sequences inherent in each class (IgG, IgM, IgA,IgD, and IgE) and each subclass (IgG1, IgG2, IgG3, and IgG4, IgA1, andIgA2).

[0076] The heavy chain of IgG (IgG1, IgG2, IgG3, and IgG4) is composedof VH, CH1 domain, hinge region, CH2 domain, and CH3 domain in thisorder from N terminus.

[0077] Similarly, the heavy chain of IgG1 is composed of VH, Cγ₁1domain, hinge region, Cγ₁2 domain, and Cγ₁3 domain in this order from Nterminus. The heavy chain of IgG2 is composed of VH, Cγ₂1 domain, hingeregion, Cγ₂2 domain, and Cγ₂3 domain in this order from N terminus. Theheavy chain of IgG3 is composed of VH, Cγ₃1 domain, hinge region, Cγ₃2domain, and Cγ₃3 domain in this order from N terminus. The heavy chainof IgG4 is composed of VH, Cγ₄1 domain, hinge region, Cγ₄2 domain, andCγ₄3 domain in this order from N terminus.

[0078] The heavy chain of IgA is composed of VH, Cα1 domain, hingeregion, Cα2 domain, and Cα3 domain in this order from N terminus.

[0079] Similarly, the heavy chain of IgA1 is composed of VH, Cα₁1domain, hinge region, Cα₁2 domain, and Cα₁3 domain in this order from Nterminus. The heavy chain of IgA2 is composed of VH, Cα₂1 domain, hingeregion, Cα₂2 domain, and Cα₂3 domain in this order from N terminus.

[0080] The heavy chain of IgD is composed of VH, Cδ1 domain, hingeregion, Cδ2 domain, and Cδ3 domain in this order from N terminus.

[0081] The heavy chain of IgM is composed of VH, Cμ1 domain, Cμ2 domain,Cμ3 domain, and Cμ4 domain in this order from N terminus and have nohinge region as seen in IgG, IgA, and IgD.

[0082] The heavy chain of IgE is composed of VH, Cε1 domain, Cε2 domain,Cε3 domain, and Cε4 domain in this order from N terminus and have nohinge region as seen in IgG, IgA, and IgD.

[0083] If, for example, IgG is treated with papain, it is cleaved at theslightly N terminal side beyond the disulfide bonds existing in thehinge region where the disulfide bonds connect the two heavy chains togenerate two homologous Fab, in which a heavy chain fragment composed ofVH and CH1 is connected with one light chain through a disulfide bond,and one Fc, in which two homologous heavy chain fragments composed ofthe hinge region, CH2 domain, and CH3 domain are connected throughdisulfide bonds (See “Immunology Illustrated”, original 2nd ed.,Nankodo, pp.65-75 (1992); and “Focus of Newest Medical Science‘Recognition Mechanism of Immune System’”, Nankodo, pp.4-7 (1991); andso on).

[0084] Namely, “a portion of a constant region of immunoglobulin heavychain” of the present invention means a portion of a constant region ofan immunoglobulin heavy chain having the structural characteristics asmentioned above, and preferably, is the constant region without C1domain, or the Fc region. Specifically, examples thereof are the regioncomposed of hinge region, C2 domain, and C3 domain from each of IgG,IgA, and IgD, and are the region composed of C2 domain, C3 domain, andC4 domain from each of IgM and IgE. A particularly preferable examplethereof is the Fc region of human-derived IgG1.

[0085] The “fusion protein” of the present invention is that composed ofthe above-described extracellular region of the protein of the presentinvention and a constant region or a portion of a constant region ofhuman immunoglobulin (Ig) heavy chain. Preferably, it is a fusionpolypeptide composed of an extracellular region of a protein of thepresent invention and a portion of a constant region of human IgG heavychain, and particularly preferably, it is a fusion polypeptide composedof an extracellular region of a protein of the present invention and theregion (Fc) composed of a hinge region, CH2 domain, and CH3 domain ofhuman IgG heavy chain. Moreover, IgG1 is preferable among IgG. Inaddition, a protein derived from human, mouse, or rat (preferably,human) is preferable as the protein of the present invention.

[0086] The fusion protein of the present invention has the advantagethat the fusion polypeptide can be purified extremely easily by usingaffinity column chromatography using the property of protein A, whichbinds specifically to the immunoglobulin fragment because the fusionpolypeptide of the present invention has a portion of a constant region(for example Fc) of an immunoglobulin such as IgG as mentioned above asa fusion partner. Moreover, since various antibodies against the Fc ofvarious immunoglobulin are available, an immunoassay for the fusionpolypeptides can be easily performed with antibodies against the Fc.

[0087] The protein, protein fragment, and fusion protein of the presentinvention can be produced not only by recombinant DNA technology asmentioned below but also by a method well known in the art such as achemical synthetic method and a cell culture method, or a modifiedmethod thereof.

[0088] The DNA of the present invention encodes the above-mentionedprotein of the present invention, and includes any nucleotide sequencethat can encode the protein of the present invention. The DNA preferablyencodes a human-derived protein of the present invention. Specificexamples of the DNA are described below.

[0089] (1) A DNA encoding a protein having the amino acid sequence ofSEQ ID NO: 4, a protein fragment composed of the extracellular region ofsaid protein, or a biological analog obtained by substituting, deleting,and/or modifying multiple amino acids, preferably 1 to 10 amino acids,particularly preferably 1 to 5 amino acids in the amino acid sequence ofsaid protein or fragment, or by inserting multiple amino acids,preferably 1 to 10 amino acids, particularly preferably 1 to 5 aminoacids, in said amino acid sequence.

[0090] (2) A DNA encoding a protein having the amino acid sequence ofSEQ ID NO: 10, a protein fragment composed of the extracellular regionof said protein, or a biological analog obtained by substituting,deleting, and/or modifying multiple amino acids, preferably 1 to 10amino acids, particularly preferably 1 to 5 amino acids, in the aminoacid sequence of said protein or fragment, or by inserting multipleamino acids, preferably 1 to 10 amino acids, particularly preferably 1to 5 amino acids, in said amino acid sequence.

[0091] (3) A DNA hybridizing with a DNA having the nucleotide sequenceof SEQ ID NO: 3 under stringent conditions.

[0092] (4) A DNA hybridizing with a DNA having the nucleotide sequenceof SEQ ID NO: 9 under stringent conditions.

[0093] Specific examples thereof are (1) a DNA having a nucleotidesequence corresponding to nucleotide residues 97 to 1419 of thenucleotide sequence of SEQ ID NO: 3, (2) a DNA comprising a nucleotidesequence corresponding to nucleotide residues 1 to 1419 of thenucleotide sequence of SEQ ID NO: 3, (3) a DNA having a nucleotidesequence corresponding to nucleotide residues 1 to 1785 of thenucleotide sequence of SEQ ID NO: 9, and (4) a DNA comprising anucleotide sequence corresponding to nucleotide residues 1 to 1785 ofthe nucleotide sequence of SEQ ID NO: 9.

[0094] The DNA of the present invention comprises either a genomic DNAor cDNA. In addition, the DNA includes any DNA composed of any codonsencoding the same amino acids.

[0095] Examples of “stringent conditions” are as follows. When a probewith 50 or more nucleotides is used and hybridization is performed in0.9% NaCl, the standard of temperature where 50% dissociation occurs(Tm) is calculated using the following formula and the temperature forhybridization can be determined according to the following formula.

Tm=82.3° C.+0.41×(G+C)%−500/n−0.61×(formamide)%

[0096] (n means the number of the nucleotide of probe).

[0097] Temperature=Tm−25° C.

[0098] In addition, when a probe with 100 or more nucleotides (G+C=40 to50%) is used, it should be considered that Tm varies as (1) and (2)mentioned below.

[0099] (1) Tm descends by about 1° C. per 1% mismatch.

[0100] (2) Tm descends by 0.6 to 0.7° C. per 1% formamide.

[0101] Accordingly, the temperature conditions for the combination ofcompletely complementary strands can be set as follows.

[0102] (A) 65 to 75° C. (formamide not added)

[0103] (B) 35 to 45° C. (in the presence of 50% formamide)

[0104] The temperature conditions for the combination of incompletelycomplementary strands can be set as follows.

[0105] (A) 45 to 55° C. (formamide not added)

[0106] (B) 35 to 42° C. (in the presence of 30% formamide)

[0107] The temperature conditions when a probe with 23 or lessnucleotides is used can be 37° C. or can be calculated using thefollowing formula. Temperature=2° C.×(the number of A+T)+4° C.×(thenumber of C+G) −5° C.

[0108] The DNA of the present invention can be a DNA obtained by anymethod. For example, the DNA includes complementary DNA (cDNA) preparedfrom mRNA, DNA prepared from genomic DNA, DNA prepared by chemicalsynthesis, DNA obtained by PCR amplification with RNA or DNA as atemplate, and DNA constructed by appropriately combining these methods.

[0109] The DNA encoding the protein of the present invention can beobtained by the usual method such as a method to clone cDNA from mRNAencoding the protein of the present invention, a method to isolategenomic DNA and then splice them, chemical synthesis and so on.

[0110] (1) cDNA can be cloned from the mRNA encoding the protein of thepresent invention by, for example, the method described below.

[0111] First, the mRNA encoding the protein of the present invention isprepared from the above-described tissues or cells expressing andproducing a cell surface molecule (polypeptide) of the presentinvention. mRNA can be prepared isolating total RNA by a known methodsuch as quanidine-thiocyanate method (Chirgwin et al., Biochemistry,Vol.18, p5294, 1979), hot phenol method, or AGPC method, and subjectingit to affinity chromatography using oligo-dT cellulose or poly-USepharose.

[0112] Then, with the mRNA obtained as a template, cDNA is synthesized,for example, by a well-known method using reverse transcriptase such asthe method of Okayama et al. (Mol. Cell. Biol. Vol.2, p.161 (1982);ibid. Vol.3, p.280 (1983)) or the method of Hoffman et al. (Gene Vol.25,p.263 (1983)), and converted into double-stranded cDNA. A cDNA libraryis prepared by transforming E. coli with plasmid vectors, phage vectors,or cosmid vectors having this cDNA or by transfecting E. coli after invitro packaging.

[0113] The plasmid vectors used in this invention are not limited aslong as they are replicated and maintained in hosts. Any phage vectorsthat can be replicated in hosts can also be used. Examples of usuallyused cloning vectors are pUC19, λgt10, λgt11, and so on. When the vectoris applied to immunological screening as mentioned below, the vectorhaving a promoter that can express a gene encoding the polypeptide ofthe present invention in a host is preferably used.

[0114] cDNA can be inserted into a plasmid by, for example, the methodof Maniatis et al. (Molecular Cloning, A Laboratory Manual, secondedition, Cold Spring Harbor Laboratory, p.1.53, 1989). cDNA can beinserted into a phage vector by, for example, the method of Hyunh et al.(DNA cloning, a practical approach, Vol.1, p.49 (1985)). These methodscan be simply performed by using a commercially available cloning kit(for example, a product from Takara Shuzo). The recombinant plasmid orphage vector thus obtained is introduced into appropriate host cellssuch as a prokaryote (for example, E. coli: HB101, DH5 α, MC1061/P3,etc.).

[0115] Examples of a method for introducing a plasmid into a host arecalcium chloride method, calcium chloride/rubidium chloride methoddescribed in Molecular Cloning, A Laboratory Manual (second edition,Cold Spring Harbor Laboratory, p.1.74 (1989)), and electroporationmethod. Phage vectors can be introduced into host cells by, for example,a method in which the phage DNAs are introduced into grown hosts afterin vitro packaging. In vitro packaging can be easily performed with acommercially available in vitro packaging kit (for example, a productfrom Stratagene or Amersham).

[0116] The cDNA encoding the protein of the present invention can beisolated from the cDNA library so prepared according to the methodmentioned above by combining general cDNA screening methods.

[0117] For example, a clone comprising the desired cDNA can be screenedby a known colony hybridization method (Crunstein et al. Proc. Natl.Acad. Sci. USA, Vol.72, p.3961 (1975)) or plaque hybridization method(Molecular Cloning, A Laboratory Manual, second edition, Cold SpringHarbor Laboratory, p.2.108 (1989)) using ³²P-labeled chemicallysynthesized oligonucleotides as probes, which are corresponding to theamino acid sequence of the polypeptide of the present invention.Alternatively, a clone having a DNA fragment encoding a specific regionwithin the polypeptide of the present invention can be screened byamplifying the region by PCR with synthetic PCR primers.

[0118] When a cDNA library prepared using a cDNA expression vector (forexample, λZAPII phage vector) is used, the desired clone can be screenedby the antigen-antibody reaction using an antibody against thepolypeptide of the present invention. A screening method using PCRmethod is preferably used when many clones are subjected to screening.

[0119] The nucleotide sequence of the DNA thus obtained can bedetermined by Maxam-Gilbert method (Maxam et al. Proc. Natl. Acad. Sci.USA, Vol.74, p.560 (1977)) or the dideoxynucleotide synthetic chaintermination method using phage M13 (Sanger et al. Proc. Natl. Acad. Sci.USA, Vol.74, pp.5463-5467 (1977)). The whole or a portion of the geneencoding the polypeptide of the present invention can be obtained byexcising the clone obtained as mentioned above with restriction enzymesand so on.

[0120] (2) The DNA encoding the polypeptide of the present invention canbe isolated from the genomic DNA derived from the cells expressing thepolypeptide of the present invention as mentioned above by the followingmethods. Such cells are solubilized preferably by SDS or proteinase K,and the DNAs are deproteinized by repeating phenol extraction. RNAs aredigested preferably with ribonuclease. The DNAs obtained are partiallydigested with appropriate restriction enzymes, and the DNA fragmentsobtained are amplified with appropriate phage or cosmid to generate alibrary. Then, clones having the desired sequence are detected, forexample, by using radioactively labeled DNA probes, and the whole or aportion of the gene encoding the protein of the present invention isobtained from the clones by excision with restriction enzyme and so on.

[0121] (3) The DNA of the present invention can also be chemicallysynthesized by the usual method, based on the nucleotide sequence of SEQID NO: 1, 3, 5, 7, 9, or 27.

[0122] The present invention also relates to a recombinant vectorcomprising the DNA encoding the protein of the present invention. Therecombinant vector of the present invention is not limited as long as itcan be replicated and maintained or can autonomously replicate invarious prokaryotic and/or eukaryotic hosts. The vector of the presentinvention includes plasmid vectors and phage vectors.

[0123] The recombinant vector can easily be prepared by ligating the DNAencoding the protein of the present invention with a vector forrecombination available in the art (plasmid DNA and bacteriophage DNA)by the usual method. Specific examples of the vectors for recombinationused are E. coli-derived plasmids such as pBR322, pBR325, pUC12, pUC13,and pUC19, yeast-derived plasmids such as pSH19 and pSH15, and Bacillussubtilis-derived plasmids such as pUB110, pTP5, and pC194. Examples ofphages are a bacteriophage such as λ phage, and an animal or insectvirus (pVL1393, Invitrogen) such as a retrovirus, vaccinia virus, andnuclear polyhedrosis virus.

[0124] An expression vector is useful for expressing the DNA encodingthe protein of the present invention and for producing the polypeptideof the present invention. The expression vector is not limited as longas it expresses the gene encoding the polypeptide of the presentinvention in various prokaryotic and/or eukaryotic host cells andproduces this protein. Examples thereof are PMAL C2, pEF-BOS (NucleicAcids Res. Vol.18, p.5322 (1990)), pME18S (Experimental Medicine:SUPPLEMENT, “Handbook of Genetic Engineering” (1992)), and so on.

[0125] When bacteria, particularly E. coli are used as host cells, anexpression vector is generally comprised of, at least, apromoter/operator region, an initiation codon, the DNA encoding theprotein of the present invention, termination codon, terminator region,and replicon.

[0126] When yeast, animal cells, or insect cells are used as hosts, anexpression vector is preferably comprised of, at least, a promoter, aninitiation codon, the DNA encoding the protein of the present invention,and a termination codon. It may also comprise the DNA encoding a signalpeptide, enhancer sequence, 5′- and 3 ′-untranslated region of the geneencoding the protein of the present invention, splicing junctions,polyadenylation site, selectable marker region, and replicon. Theexpression vector may also contain, if required, a gene for geneamplification (marker) that is usually used.

[0127] A promoter/operator region to express the polypeptide of thepresent invention in bacteria comprises a promoter, an operator, and aShine-Dalgarno (SD) sequence (for example, AAGG). For example, when thehost is Escherichia, it preferably comprises Trp promoter, lac promoter,recA promoter, λ PL promoter, lpp promoter, tac promoter, or the like.Examples of a promoter to express the polypeptide of the presentinvention in yeast are PH05 promoter, PGK promoter, GAP promoter, ADHpromoter, and so on. When the host is Bacillus, examples thereof areSL01 promoter, SP02 promoter, penP promoter and so on. When the host isa eukaryotic cell such as a mammalian cell, examples thereof areSV40-derived promoter, retrovirus promoter, heat shock promoter, and soon, and preferably Sv-40 and retrovirus-derived one. As a matter ofcourse, the promoter is not limited to the above examples. In addition,to use an enhancer is effective for expression.

[0128] A preferable initiation codon is, for example, a methionine codon(ATG).

[0129] The commonly used termination codon (for example, TAG, TGA, TAA,and so on) is illustrated as a termination codon.

[0130] Usually used natural or synthetic terminators are used as aterminator region.

[0131] A replicon means a DNA capable of replicating the whole DNAsequence in host cells, and includes a natural plasmid, an artificiallymodified plasmid (DNA fragment prepared from a natural plasmid), asynthetic plasmid, and so on. Examples of a preferable plasmids arepBR322 or its artificial derivatives (DNA fragment obtained by treatingpBR322 with appropriate restriction enzymes) for E. coli, yeast2 μplasmid or yeast chromosomal DNA for yeast, and pRSVneo ATCC 37198,pSV2dhfr ATCC 37145, pdBPV-MMTneo ATCC 37224, pSV2neo ATCC 37149, etc.for mammalian cells.

[0132] An enhancer sequence, polyadenylation site, and splicing junctionthat are usually used in the art, such as those derived from SV40 can bealso used.

[0133] A selectable marker usually used can be used according to theusual method. Examples thereof are resistance genes for antibiotics,such as tetracycline, neomycin, ampicillin, or kanamycin, and thymidinekinase gene.

[0134] Examples of a gene for gene amplification are dihydrofolatereductase (DHFR) gene, thymidine kinase gene, neomycin resistance gene,glutamate synthase gene, adenosine deaminase gene, ornithinedecarboxylase gene, hygromycin-B-phophotransferase gene, aspartatetranscarbamylase gene, etc.

[0135] The expression vector of the present invention can be prepared bycontinuously and circularly linking at least the above-mentionedpromoter, initiation codon, DNA (gene) encoding the polypeptide of thepresent invention, termination codon, and terminator region, to anappropriate replicon. If desired, appropriate DNA fragments (forexample, linkers, restriction sites generated with other restrictionenzyme), can be used by the usual method such as digestion with arestriction enzyme or ligation using T4 DNA ligase.

[0136] Transformants of the present invention can be prepared byintroducing the expression vector mentioned above into host cells.

[0137] Host cells used in the present invention are not limited as longas they are compatible with an expression vector mentioned above and canbe transformed. Examples thereof are various cells such as natural cellsor artificially established recombinant cells usually used in technicalfield of the present invention (for example, bacteria (Escherichia andBacillus), yeast (Saccharomyces, Pichia, etc.), animal cells, or insectcells.

[0138]E. coli or animal cells are preferably used. Specific examples areE. coli (DH5α, TB1, HB101, etc.), mouse-derived cells (COP, L, C127,Sp2/0, NS-1, NIH 3T3, etc.), rat-derived cells, hamster-derived cells(BHK, CHO, etc.), monkey-derived cells (COS1, COS3, COS7, CV1, Velo,etc.), and human-derived cells (Hela, diploid fibroblast-derived cells,HEK293, myeloma, Namalwa, etc.).

[0139] An expression vector can be introduced (transformed (transduced))into host cells by known method.

[0140] Transformation can be performed, for example, according to themethod of Cohen et al. (Proc. Natl. Acad. Sci. USA, Vol.69, p.2110(1972)), protoplast method (Mol. Gen. Genet., Vol.168, p.111 (1979)), orcompetent method (J. Mol. Biol., Vol.56, p.209 (1971)) when the hostsare bacteria (E. coli, Bacillus subtilis, etc.), the method of Hinnen etal. (Proc. Natl. Acad. Sci. USA, Vol.75, p.1927 (1978)), or lithiummethod (J. Bacteriol., Vol.153, p.163 (1983)) when the host isSaccharomyces cerevisiae, the method of Graham (Virology, Vol.52, p.456(1973)) when the hosts are animal cells, and the method of Summers etal. (Mol. Cell. Biol., Vol.3, pp.2156-2165 (1983)) when the hosts areinsect cells.

[0141] The protein of the present invention can be produced bycultivating transformants (in the following this term includestransductants) comprising an expression vector prepared as mentionedabove in nutrient media.

[0142] The nutrient media preferably comprise carbon source, inorganicnitrogen source, or organic nitrogen source necessary for the growth ofhost cells (transformants). Examples of the carbon source are glucose,dextran, soluble starch, and sucrose, and examples of the inorganic ororganic nitrogen source are ammonium salts, nitrates, amino acids, cornsteep liquor, peptone, casein, meet extract, soy bean cake, and potatoextract. If desired, they may comprise other nutrients (for example, aninorganic salt (for example, calcium chloride, sodiumdihydrogenphosphate, and magnesium chloride), vitamins, antibiotics (forexample, tetracycline, neomycin, ampicillin, kanamycin, etc.).

[0143] Cultivation is performed by a method known in the art.Cultivation conditions such as temperature, pH of the media, andcultivation time are selected appropriately so that the protein of thepresent invention is overproduced.

[0144] Specific media and cultivation conditions used depending on hostcells are illustrated below, but are not limited thereto.

[0145] When the hosts are bacteria, actinomycetes, yeasts, filamentousfungi, liquid media comprising the nutrient source mentioned above areappropriate. The media with pH 5 to 8 are preferably used.

[0146] When the host is E. coli, examples of preferable media are LBmedia, and M9 media (Miller et al. Exp. Mol. Genet., Cold Spring HarborLaboratory, p.431 (1972)). Using these media, cultivation can beperformed usually at 14 to 43° C. for about 3 to 24 hours with aerationand stirring, if necessary.

[0147] When the host is Bacillus, cultivation can be performed usuallyat 30 to 40° C. for about 16 to 96 hours with aeration and stirring, ifnecessary.

[0148] When the host is yeast, examples of media are Burkholder minimalmedia (Bostian, Proc. Natl. Acad. Sci. USA, Vol.77, p.4505 (1980)). ThepH of the media is preferably 5 to 8. Cultivation can be performedusually at 20 to 35° C. for about 14to 144 hours with aeration andstirring, if necessary.

[0149] When the host is an animal cell, examples of media are MEM mediacontaining about 5 to 20% fetal bovine serum (Science, Vol.122, p.501(1952)), DMEM media (Virology, Vol.8, p.396 (1959)), RPMI1640 media (J.Am. Med. Assoc., Vol.199, p.519 (1967)), and 199 media (Proc. Soc. Exp.Biol. Med., Vol.73, p.1 (1950)). The pH of the media is preferably about6 to 8. Cultivation can be performed usually at about 30 to 40° C. forabout 15 to 72 hours with aeration and stirring, if necessary.

[0150] When the host is an insect cell, an example of media is Grace'smedia containing fetal bovine serum (Proc. Natl. Acad. Sci. USA, Vol.82,p.8404 (1985)). The pH thereof is preferably about 5to 8. Cultivationcan be performed usually at about 20 to 40° C. for 15 to 100 hours withaeration and stirring, if necessary.

[0151] The protein of the present invention can be produced as atransmembrane protein by cultivating transformants as mentioned above,in particular animal cells to overexpress the protein of the presentinvention on the surface of the cells. The protein of the presentinvention can be produced as a soluble protein fragment such as anextracellular region protein fragment by preparing the transformants asmentioned above using the DNA encoding the extracellular region and bycultivating the transformants to allow them to secrete the solublepolypeptide into the culture supernatant.

[0152] Namely, a culture filtrate (supernatant) is obtained by themethod such as filtration or centrifugation of the obtained culture, andthe protein of the present invention is purified and isolated from theculture filtrate by the usual method commonly used in order to purifyand isolate a natural or synthetic protein.

[0153] Examples of the isolation and purification method are a methodutilizing solubility, such as salting out and solvent precipitationmethod, a method utilizing the difference in molecular weight, such asdialysis, ultrafiltration, gel filtration, and sodium dodecylsulfate-polyacrylamide gel electrophoresis, a method utilizing charges,such as ion exchange chromatography and hydroxylapatite chromatography,a method utilizing specific affinity, such as affinity chromatography, amethod utilizing the difference in hydrophobicity, such as reverse phasehigh performance liquid chromatography, and a method utilizing thedifference in isoelectric point, such as isoelectric focusing.

[0154] When the protein of the present invention exists in the periplasmor cytoplasm of cultured transformants, first, the fungus bodies orcells are harvested by the usual method such as filtration orcentrifugation and suspended in appropriate buffer. After the cell walland/or cell membrane of the cells and so on are disrupted by the methodsuch as lysis with sonication, lysozyme, and freeze-thawing, themembrane fraction comprising the protein of the present invention isobtained by the method such as centrifugation or filtration. Themembrane fraction is solubilized with a detergent such as Triton-X100 toobtain the crude extract. Finally, the polypeptide or the polypeptidefragment is isolated and purified from the crude extract by the usualmethod as illustrated above.

[0155] The “transgenic mouse” of the present invention is a transgenicmouse wherein the DNA (cDNA or genomic DNA) prepared as mentioned aboveencoding the protein of the present invention derived from animalsexcept mice (non-self protein) have been integrated into its endogenouslocus of the mouse. The transgenic mouse expresses the non-self proteinand secretes the protein into its body.

[0156] The transgenic mouse can be prepared according to the method asusually used for producing a transgenic animal (for example, see “NewestManual of Animal Cell Experiment”, LIC press, Chapter 7, pp.361-408,(1990)).

[0157] Specifically, for example, embryonic stem cells (ES cells)obtained from normal mouse blastocysts are transformed with anexpression vector in which the gene encoding human-derived polypeptideof the present invention (i.e. “human JTT-1 antigen”) has been operablyinserted. ES cells in which the gene encoding the human-derivedpolypeptide of the present invention has been integrated into theendogenous gene are screened by the usual method. Then, the ES cellsscreened are microinjected into a fertilized egg obtained from anothernormal mouse (blastocyst) (Proc. Natl. Acad. Sci. USA, Vol.77, No.12,pp.7380-7384 (1980); U.S. Pat. No. 4,873,191). The blastocyst istransplanted into the uterus of another normal mouse as the fostermother. Then, founder mice (progeny mice) are born from the fostermother mouse. By mating the founder mice with normal mice, heterogeneictransgenic mice are obtained. By mating the heterogeneic transgenic micewith each other, homogeneic transgenic mice are obtained according toMendel's laws.

[0158] “Knockout mouse” of the present invention is a mouse wherein theendogenous gene encoding the mouse-derived protein of the presentinvention has been knocked out (inactivated). It can be prepared, forexample, by positive-negative selection method in which homologousrecombination is applied (U.S. Pat. No. 5,464,764; U.S. Pat. No.5,487,992; U.S. Pat. No. 5,627,059; Proc. Natl. Acad. Sci. USA, Vol.86,pp.8932-8935 (1989); Nature, Vol.342, pp.435-438 (1989); etc.).

[0159] The “antibody” of the present invention can be a polyclonalantibody (antiserum) or a monoclonal antibody, and preferably amonoclonal antibody.

[0160] Specifically, it is an antibody reactive to (against, which bindsto) the above-mentioned protein or its fragment of the presentinvention.

[0161] The antibody of the present invention can be natural antibodiesobtained by immunizing mammals such as mice, rats, hamsters, guineapigs, and rabbits with an immunogen (antigen), such as the protein ofthe present invention (natural, recombinant, or synthetic ones), cellsexpressing the protein of the present invention, or transformantsoverexpressing the designed protein on the surface thereof preparedusing recombinant DNA technology as described above on the cell surface.The antibody of the present invention also includes chimeric antibodiesand humanized antibodies (CDR-grafted antibodies) that can be producedby recombinant DNA technology, and human antibodies that can be producedusing human antibody-producing transgenic animals.

[0162] The monoclonal antibody includes those having any one isotype ofIgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable.

[0163] The polyclonal antibody (antisera) or monoclonal antibody of thepresent invention can be produced by the known methods. Namely, amammal, preferably, a mouse, rat, hamster, guinea pig, rabbit, cat, dog,pig, goat, horse, or cattle, or more preferably, a mouse, rat, hamster,guinea pig, or rabbit is immunized, for example, with an immunogen(antigen) mentioned above with Freund's adjuvant, if necessary. Thepolyclonal antibody can be obtained from the antiserum obtained from theanimal so immunized. In addition, the monoclonal antibodies are producedas follows. Hybridomas are prepared from the antibody-producing cellsobtaind from the animal so immunized and myeloma cells that are notcapable of producing autoantibodies. The hybridomas are cloned, andclones producing the monoclonal antibodies showing the specific affinityto the antigen used for immunizing the mammal are screened.

[0164] Specifically, the monoclonal antibody can be produced as follows.Immunizations are performed by injecting or implanting once or severaltimes the protein of the present invention, cells expressing the proteinand so on as mentioned above as an immunogen, if necessary, withFreund's adjuvant, subcutaneously, intramuscularly, intravenously,through the footpad, or intraperitoneally into a mouse, rat, hamster,guinea pig, or rabbit, preferably a mouse, rat, or hamster (including atransgenic animal generated so as to produce antibodies derived fromanother animal such as the transgenic mouse producing human antibody).Usually, immunizations are performed once to four times every one tofourteen days after the first immunization. Antibody-producing cells areobtained from the mammal so immunized in about one to five days afterthe last immunization.

[0165] Hybridomas that secrete a monoclonal antibody can be prepared bythe method of Köhler and Milstein (Nature, Vol.256, pp.495-497 (1975))and by its modified method. Namely, hybridomas are prepared by fusingantibody-producing cells contained in a spleen, lymph node, bone marrow,or tonsil obtained from the mammal immunized as mentioned above,preferably a spleen, with myelomas without autoantibody-producingability, which are derived from, preferably, a mammal such as a mouse,rat, guinea pig, hamster, rabbit, or human, or more preferably, a mouse,rat, or human.

[0166] For example, mouse-derived myeloma P3/X63-AG8.653 (653),P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1), SP2/0-Ag14 (Sp2/0, Sp2),PAI, F0, or BW5147, rat-derived myeloma 210RCY3-Ag.2.3., orhuman-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR,D1R11, or CEM-T15 can be used as a myeloma used for the cell fusion.

[0167] Hybridoma clones producing monoclonal antibodies can be screenedby cultivating hybridomas, for example, in microtiter plates and bymeasuring the reactivity of the culture supernatant in the well in whichhybridoma growth is observed, to the immunogen used for the immunizationmentioned above, for example, by enzyme immunoassay such as RIA andELISA.

[0168] The monoclonal antibodies can be produced from hybridomas bycultivating the hybridomas in vitro or in vivo such as in the ascitesfluid of a mouse, rat, guinea pig, hamster, or rabbit, preferably amouse or rat, more preferably mouse and isolating the antibodies fromthe resulting the culture supernatant or ascites fluid of a mammal.

[0169] Cultivating hybridomas in vitro can be performed depending on theproperty of cells to be cultured, on the object of a test study, and onthe various conditions of a cultivating method, by using known nutrientmedia or any nutrient media derived from known basal media for growing,maintaining, and storing the hybridomas to produce monoclonal antibodiesin culture supernatant.

[0170] Examples of basal media are low calcium concentration media suchas Ham'F12 medium, MCDB153 medium, or low calcium concentration MEMmedium, and high calcium concentration media such as MCDB104 medium, MEMmedium, D-MEM medium, RPM11640 medium, ASF104 medium, or RD medium. Thebasal media can contain, for example, sera, hormones, cytokines, and/orvarious inorganic or organic substances depending on the objective.

[0171] Monoclonal antibodies can be isolated and purified from theculture supernatant or ascites fluid mentioned above by saturatedammonium sulfate precipitation, euglobulin precipitation method, caproicacid method, caprylic acid method, ion exchange chromatography (DEAE orDE52), affinity chromatography using anti-immunoglobulin column orprotein A column.

[0172] The “chimeric antibody” of the present invention is a monoclonalantibody prepared by genetic engineering, and specifically means achimeric antibody such as mouse/human chimeric monoclonal antibody whosevariable regions or the other regions are derived from mouseimmunoglobulin and whose constant regions are derived from humanimmunoglobulin.

[0173] The constant region derived from human immunoglobulin has theamino acid sequence inherent in each isotype such as IgG, IgM, IgA, IgD,and IgE. The constant region of the recombinant chimeric monoclonalantibody of the present invention can be that of human immunoglobulinbelonging to any isotype. Preferably, it is the constant region of humanIgG.

[0174] The chimeric monoclonal antibody of the present invention can beproduced, for example, as follows. Needless to say, the productionmethod is not limited thereto.

[0175] A mouse/human chimeric monoclonal antibody can be prepared,referring to Experimental Medicine: SUPPLEMENT, Vol.1.6, No.10 (1988);and examined published Japanese patent application (JP-B) No.Hei3-73280. Namely, it can be prepared by operably inserting CH gene (Cgene encoding the constant region of H chain) obtained from the DNAencoding human immunoglobulin downstream of active VH genes (rearrangedVDJ gene encoding the variable region of H chain) obtained from the DNAencoding a mouse monoclonal antibody isolated from the hybridomaproducing the mouse monoclonal antibody, and CL gene (C gene encodingthe constant region of L chain) obtained from the DNA encoding humanimmunoglobulin downstream of active VL genes (rearranged VJ geneencoding the variable region of L chain) obtained from the DNA encodingthe mouse monoclonal antibody isolated from the hybridoma, into the sameor different vectors so as for them to be expressed, following bytransforming host cells with the expression vector, and then bycultivating the transformants.

[0176] Specifically, DNAs are first extracted from mouse monoclonalantibody-producing hybridomas by the usual method, digested withappropriate restriction enzymes (for example, EcoRI and HindIII),electrophoresed (using, for example, 0.7% agarose gel), and analyzed bySouthern blotting. After an electrophoresed gel is stained, for example,with ethidium bromide and photographed, the gel is given with markerpositions, washed twice with water, and soaked in 0.25 M HCl for 15minutes. Then, the gel is soaked in 0.4 N NaOH solution for 10 minuteswith gently stirring. The DNAs are transferred to a filter for 4 hoursby the usual method. The filter is recovered and washed twice with2×SSC. After the filter is sufficiently dried, it is baked at 75° C. for3 hours. After baking, the filter is treated with 0.1×SSC/0.1% SDS at65° C. for 30 minutes. Then, it is soaked in 3×SSC/0.1% SDS. The filterobtained is treated with prehybridization solution in a plastic bag at65° C. for 3 to 4 hours.

[0177] Next, ³²P-labeled probe DNA and hybridization solution are addedto the bag and reacted at 65° C. about 12 hours. After hybridization,the filter is washed under appropriate salt concentration, reactiontemperature, and time (for example, 2×SSC-0.1% SDS, room temperature, 10minutes). The filter is put into a plastic bag with a little 2×SSC, andsubjected to autoradiography after the bag is sealed.

[0178] Rearranged VDJ gene and VJ gene encoding H chain and L chain of amouse monoclonal antibody are identified by Southern blotting mentionedabove. The region comprising the identified DNA fragment is fractionedby sucrose density gradient centrifugation and inserted into a phagevector (for example, Charon 4A, Charon 28, λEMBL3, λEMBL4, etc.). E.coli (for example, LE392, NM539, etc.) is transformed with the phagevector to generate a genomic library. The genomic library is screened byplaque hybridization such as Benton-Davis method (Science, Vol.196,pp.180-182 (1977)) using appropriate probes (H chain J gene, L chain (κ)J gene, etc.) to obtain positive clones comprising rearranged VDJ geneor VJ gene. By making the restriction map and determining the nucleotidesequence of the clones obtained, it is confirmed that genes comprisingthe desired, rearranged VH (VDJ) gene or VL (VJ) gene are obtained.

[0179] Separately, human CH gene and human CL gene used forchimerization are isolated. For example, when a chimeric antibody withhuman IgGl is produced, Cγ1 gene as a CH gene, and Cκ gene as a CL gene,are isolated. These genes can be isolated from human genomic librarywith mouse Cγ1 gene and mouse Cκ gene, corresponding to human Cγ1 geneand human C κ gene, respectively, as probes, taking advantage of highhomology between the nucleotide sequences of mouse immunoglobulin geneand that of human immunoglobulin gene.

[0180] Specifically, DNA fragments comprising human Cκ gene and anenhancer region are isolated from human λ Charon 4A HaeIII-AluI genomiclibrary (Cell, Vol.15, pp.1157-1174 (1978)), for example, with a 3 kbHindIII-BamHI fragment of clone Ig146 (Proc. Natl. Acad. Sci. USA,Vol.75, pp.4709-4713 (1978)) and a 6.8 kb EcoRI fragment of clone MEP10(Proc. Natl. Acad. Sci. USA, Vol.78, pp.474-478 (1981)) as probes. Inaddition, for example, after human fetal hepatocyte DNA is digested withHindIII and fractioned by agarose gel electrophoresis, a 5.9 kb fragmentis inserted into λ788 and then human Cγ1 gene is isolated with theprobes mentioned above.

[0181] Using mouse VH gene, mouse VL gene, human CH gene, and human CLgene so obtained, and taking promoter region and enhancer region intoconsideration, human CH gene is inserted downstream mouse VH gene andhuman CL gene is inserted downstream mouse VL gene into an expressionvector such as pSV2gpt or pSV2neo with appropriate restriction enzymesand DNA ligase by the usual method. In this case, chimeric genes ofmouse VH gene/human CH gene and mouse VL gene/human CL gene can berespectively inserted in the same expression vector or in differentexpression vectors.

[0182] Chimeric gene-inserted expression vector(s) thus prepared areintroduced into myelomas that do not produce antibodies, for example,P3X63•Ag8•653 cells or SP210 cells by protoplast fusion method,DEAE-dextran method, calcium phosphate method, or electroporationmethod. The transformants are screened by cultivating in mediacontaining a drug corresponding to the drug resistance gene insertedinto the expression vector and, then, cells producing desired chimericmonoclonal antibodies are obtained.

[0183] Desired chimeric monoclonal antibodies are obtained from theculture supernatant of antibody-producing cells thus screened.

[0184] The “humanized antibody (CDR-grafted antibody)” of the presentinvention is a monoclonal antibody prepared by genetic engineering andspecifically means a humanized monoclonal antibody wherein a portion orthe whole of the complementarity determining regions of thehypervariable region are derived from the complementarity determiningregions of the hypervariable region from a mouse monoclonal antibody,the framework regions of the variable region are derived from theframework regions of the variable region from human immunoglobulin, andthe constant region is derived from human a constant region fromimmunoglobulin.

[0185] The complementarity determining regions of the hypervariableregion exists in the hypervariable region in the variable region of anantibody and means three regions which directly and complementary bindsto an antigen (complementarity-determining residues, CDR1, CDR2, andCDR3). The framework regions of the variable region means fourcomparatively conserved regions lying upstream, downstream or betweenthe three complementarity determining regions (framework region, FR1,FR2, FR3, and FR4).

[0186] In other words, a humanized monoclonal antibody means that inwhich the whole region except a portion or the whole of thecomplementarity determining regions of the hypervariable region of anonhuman mammal-derived monoclonal antibody have been replaced withtheir corresponding regions derived from human immunoglobulin.

[0187] The constant region derived from human immunoglobulin has theamino acid sequence inherent in each isotype such as IgG (IgG1, IgG2,IgG3, IgG4), IgM, IgA, IgD, and IgE. The constant region of a humanizedmonoclonal antibody in the present invention can be that from humanimmunoglobulin belonging to any isotype. Preferably, it is the constantregion of human IgG. The framework regions of the constant regionderived from human immunoglobulin are not particularly limited.

[0188] The humanized monoclonal antibody of the present invention can beproduced, for example, as follows. Needless to say, the productionmethod is not limited thereto.

[0189] For example, a recombinant humanized monoclonal antibody derivedfrom mouse monoclonal antibody can be prepared by genetic engineering,referring to unexamined Japanese patent publication (JP-WA) No. Hei4-506458 and unexamined Japanese patent publication (JP-A) No. Sho62-296890. Namely, at least one mouse H chain CDR gene and at least onemouse L chain CDR gene corresponding to the mouse H chain CDR gene areisolated from hybridomas producing mouse monoclonal antibody, and humanH chain gene encoding the whole regions except human H chain CDRcorresponding to mouse H chain CDR mentioned above and human L chaingene encoding the whole region except human L chain CDR correspond tomouse L chain CDR mentioned above are isolated from human immunoglobulingenes.

[0190] The mouse H chain CDR gene(s) and the human H chain gene(s) soisolated are operably inserted into an appropriate vector so that theycan be expressed. Similarly, the mouse L chain CDR gene(s) and the humanL chain gene(s) are operably inserted into another appropriate vector sothat they can be expressed. Alternatively, the mouse H chain CDRgene(s)/human H chain gene(s) and mouse L chain CDR gene(s)/human Lchain gene(s) can be operably inserted into the same expression vectorso that they can be expressed. Host cells are transformed with theexpression vector thus prepared to obtain transformants producinghumanized monoclonal antibody. By cultivating the transformants, desiredhumanized monoclonal antibody is obtained from culture supernatant.

[0191] The “human monoclonal antibody” of the present invention isimmunoglobulin in which the entire regions comprising the variable andconstant region of H chain, and the variable and constant region of Lchain constituting immunoglobulin are derived from the gene encodinghuman immunoglobulin.

[0192] The human antibody can be produced in the same way as theproduction method of polyclonal or monoclonal antibodies mentioned aboveby immunizing, with an antigen, a transgenic animal which for example,at least human immunoglobulin gene(s) have been integrated into thelocus of a non-human mammal such as a mouse by the usual method. Forexample, a transgenic mouse producing human antibodies is prepared bythe methods described in Nature Genetics, Vol.15, pp.146-156 (1997);Nature Genetics, Vol.7, pp.13-21 (1994); JP-WA Nos. Hei4-504365,International patent publication No. WO94/25585; Nikkei Science, No.6,pp.40-50 (1995); Nature, Vol.368, pp.856-859 (1994);and JP-WA No. Hei6-500233.

[0193] The “portion of an antibody” used in the present invention meansa partial region of the antibody, preferably monoclonal antibody of thepresent invention as mentioned above, and specifically, means F(ab′)₂,Fab′, Fab, Fv (variable fragment of antbody), sFv, dsfv (disulfidestabilized Fv),or dAb (single domain antibody) (Exp. Opin. Ther.Patents, Vol.6, No.5, pp.441-456 (1996)). “F(ab′)2” and “Fab′” can beproduced by treating immunoglobulin (monoclonal antibody) with aprotease such as pepsin and papain, and means an antibody fragmentgenerated by digesting immunoglobulin near the disulfide bonds existingbetween the hinge regions in each of the two H chains. For example,papain cleaves IgG upstream of the disulfide bonds existing between thehinge regions in each of the two H chains to generate two homologousantibody fragments in which an L chain composed of VL (L chain variableregion) and CL (L chain constant region), and an H chain fragmentcomposed of VH (H chain variable region) and CHγ1 (γ1 region in theconstant region of H chain) are connected at their C terminal regionsthrough a disulfide bond. Each of such two homologous antibody fragmentsis called Fab′. Pepsin also cleaves IgG downstream of the disulfidebonds existing between the hinge regions in each of the two H chains togenerate an antibody fragment slightly larger than the fragment in whichthe two above-mentioned Fab′ are connected at the hinge region. Thisantibody fragment is called F(ab′)₂.

[0194] The “pharmaceutical composition” of the present inventioncomprises any one of the protein, protein fragment, fusion proteinantibody, or portion of an antibody of the present invention as definedabove; and a pharmaceutically acceptable carrier.

[0195] The “pharmaceutically acceptable carrier” includes a excipieut, adiluent, an expander, a decomposition agent, a stabilizer, apreservative, a buffer, an emulsifier, an aromatic, a colorant, asweetener, a viscosity increasing agent, a flavor, a solubilityincreasing agent, or other additives. Using one or more of suchcarriers, a pharmaceutical composition can be fomulated into tablets,pills, powders, granules, injections, solutions, capsules, troches,elixirs, suspensions, emulsions, or syrups. The pharmaceuticalcomposition can be administered orally or parenterally. Other forms forparenteral administration include a solution for external application,suppository for rectal administration, and pessary, prescribed by theusual method, which comprises one or more active ingredient.

[0196] The dosage can vary depending on the age, sex, weight, andsymptom of a patient, effect of treatment, administration route, periodof treatment, or the kind of active ingredient (polypeptide or antibodymentioned above) contained in the pharmaceutical composition. Usually,the pharmaceutical composition can be administered to an adult in a doseof 10 μg to 1000 mg (or 10 μg to 500 mg) per one administration.Depending on various conditions, the dosage less than that mentionedabove may be sufficient in some cases, and the dosage more than thatmentioned above may be necessary in other cases.

[0197] In particular, the injection can be produced by dissolving orsuspending the antibody in a non-toxic, pharmaceutically acceptablecarrier such as physiological saline or commercially available distilledwater for injection with adjusting a concentration to 0.1 μg antibody/mlcarrier to 10 mg antibody/ml carrier. The injection thus produced can beadministered to a human patient in need of treatment in a dose of 1 μgto 100 mg/kg body weight, preferably 50 μg to 50 mg/kg body weight onceor more times a day. Examples of administration route are medicallyappropriate administration routes such as intravenous injection,subcutaneous injection, intradermal injection, intramuscular injection,or intraperitoneal injection, preferably intravenous injection.

[0198] The injection can also be prepared into a non-aqueous diluent(for example, propylene glycol, polyethylene glycol, vegetable oil suchas olive oil, and alcohol such as ethanol), suspension, or emulsion.

[0199] The injection can be sterilized by filtration with abacteria-non-penetrated filter, by mixing bacteriocide, or byirradiation. The injection can be produced in the form that is preparedupon use. Namely, it is freeze-dried to be a sterile solid composition,and can be dissolved in sterile distilled water for injection or anothersolvent before use.

[0200] The pharmaceutical composition of the present invention can beused to treat or prevent arteriosclerosis and restenosis after thetreatment of artery occlusion, such as PTCA.

BRIEF DESCRIPTION OF THE DRAWINGS

[0201]FIG. 1 is a photograph showing an electrophoresis image of rabbitBA2303 cDNA samples obtained by RT-PCR.

[0202] The numerals indicate days from the exfoliation of the arteryendothelium using a balloon catheter to the removal of the artery; thus,the figure shows the time course of the cDNA expression.

[0203]FIG. 2 is a photograph showing an electrophoresis image of rabbitBA0306 cDNA samples obtained by RT-PCR.

[0204] The numerals indicate days from the exfoliation of the arteryendothelium using a balloon catheter to the removal of the artery; thus,the figure shows the time course of the cDNA expression.

[0205]FIG. 3 shows a plot of the hydrophobicity and hydrophilicity ofthe amino acid residues composing rabbit BA2303 protein.

[0206]FIG. 4 shows a plot of the hydrophobicity and hydrophilicity ofthe amino acid residues composing human BA0306 protein.

[0207]FIG. 5 shows a plot of the hydrophobicity and hydrophilicity ofthe amino acid residues composing human BA2303 protein.

[0208]FIG. 6 is a photograph showing the result of Northern blotanalysis of the expression of human BA2303 mRNA in various humantissues.

[0209]FIG. 7 is a photograph showing the result of Northern blotanalysis of the expression of human BA0306 mRNA in various humantissues.

[0210]FIG. 8 shows a plot of the hydrophobicity and hydrophilicity ofthe amino acid residues composing mouse BA2303 protein.

[0211]FIG. 9 shows the sequence homology at the amino acid level betweenBA2303 proteins from rabbit, human, and mouse.

[0212]FIG. 10 shows the sequence homology at the amino acid levelbetween BA0306 proteins from rabbit, human, and mouse.

[0213]FIG. 11 schematically shows the structures of mouse genomic DNAcontaining exons that encode mouse BA2303 protein, and of the targetingvector for knockout mice generation.

[0214]FIG. 12 schematically shows the structures of mouse genomic DNAcontaining exons that encode mouse BA0306 protein, and of the targetingvector for knockout mice generation.

BEST MODE FOR IMPLEMENTING THE INVENTION

[0215] The present invention is illustrated in detail below withreference to examples, but is not to be construed as being limitedthereto.

EXAMPLE 1 Generation of a Rabbit Model Whose Aortal Endothelium isDetached by PTCA

[0216] According to the method described in “Protocols in CirculationResearch” (Jikken-Igaku Zoukan (1996) Vol.14 (12), 87), a ballooncatheter was inserted into the thoracic artery of Japanese white rabbitsby surgical operation and was inflated to perform PTCA. The arteryincluding the operation site was removed at certain periods from day 1to six months after PTCA.

EXAMPLE 2 Preparation of Total RNA from Removed Aortae

[0217] The aorta was removed at 1, 2, 4, 7, 14, 23, 30, 54, 112, and 137days after PTCA, and total RNA was prepared from the aortae by thestandard method using the TRIZOL reagent (GIBCO BRL).

[0218] Also, the aorta was removed from a normal Japanese white rabbit,which was not subjected to PTCA, and total RNA was prepared asdescribed.

EXAMPLE 3 cDNA Synthesis

[0219] Total RNAs (each 2 μl, 1 μg/ml) sampled with the passage of timeor mRNA samples (each 2 μl, 0.5 μg/ml), which were obtained in Example2, were dissolved in diethyl pirocarbonate (DEPC)-treated distilledwater (8 μl). Anchor primer (GT15MA, 1 μl, 25 pmol/μl) was added to makethe total volume 10μl, and the mixture was then incubated 5 min at 65°C. The samples were placed on ice immediately after completion of theincubation.

[0220] Then, 5×first strand buffer (4 μl, composition: 0.25 M Tris-HCl(pH 7.5), 0.375 M KCl, 0.05 M DTT, 0.015 M MgCl₂), 0.1 M DTT (2 μl), 250μM dNTP (1 μl), distilled water (1 μl), and reverse transcriptase(Superscript, GIBCO BRL, 1 μl, 200 U/μl) were added to make the totalvolume20 μl. cDNA was synthesized by incubating the reaction mixture for1 hr at 42° C., and then DEPC-treated water (30 μl) was added to makethe final volume 50 μl.

EXAMPLE 4 Analysis of the Time Course of Gene Expression

[0221] The time course of gene expression after PTCA was analyzed by thestandard method using differential display (Nucleic Acid Research (1993)Vol. 21(18), 4272-4280; Science (1992) Vol. 257, 967-971), and RT-PCR(reverse transcription-polymerase chain reaction; “PCR and itsApplication” (Jikken-Igaku Zoukan (1990) Vol. 8(9); “Gene AmplificationPCR Method/Principles and Novel Applications” Kyoritsu-Syuppan (1992)).

[0222] One hundred-fold dilution of the cDNA samples (each time point)which were prepared in Example 3 was used as a template for PCR indifferential display. Fifty fold dilution was used for cDNA samples thatwere synthesized from mRNA (each time point).

[0223] The template cDNA (each 2 μl) was mixed with distilled water(10.75 μl), 10×EX Taq buffer (2 μl), 25 μM dNTP (1.5 μl), arbitraryprimer (sequence: GATCAATCGC, 1 μl, 25pmol/μl), anchor primer (1 μl, 25pmol/μl), EX Taq DNA polymerase (0.25 μl), and α35S-dATP (1.5 μl, 10mCi/ml, Amersham) to make the total volume 20 μl. PCR was carried outwith a cycle of 95° C. for 3 min, 40° C. for 5 min, 72° C. for 5 min; 40cycles of 95° C. for 30 sec, 40° C. for 2 min, 72° C. for 1 min; and astep of 72° C. for 5 min, and then the samples were kept at 4° C.

[0224] Each of the resulting PCR products was mixed with stop buffer (5μl, composition: formamide (30 ml), xylenecyanol (30 mg), bromophenolblue (10 mg), 0.5 M EDTA (200 μl, (pH 8.0)), and then, 3.5 μl of eachresulting mixture was subjected to sequence gel electrophoresis on a 6%acrylamide gel (composition (in 500 ml total): urea (240 g), 10×TBE (50ml), 40% acrylamide (75 ml, a mixture of 38% monoacrylamide and 2%bisacrylamide)). The result showed that there were two bands whoseexpression was changed in the time course.

[0225] Both bands were excised from the gel, and two DNA fragmentscontaining the nucleotide sequences described in SEQ ID NO: 11 (178 bp)and SEQ ID NO: 12 (167 bp) were isolated according to the standardmethod (“Gene Engineering Handbook” Jikken-Igaku, Yodosya (1992)). Thefragments were named as BA2303 (SEQ ID NO: 11), and BA0306 (SEQ ID NO:12), respectively. To confirm the expression of the DNAs containing thetwo fragments in the time course, RT-PCR was performed using cDNAsamples obtained in Example 3 (each time point) as a template.

[0226] For amplification of BA2303, synthetic DNA fragments described inSEQ ID NO: 13 and SEQ ID NO: 14 were used as forward and reverseprimers, respectively.

[0227] For amplification of BA0306, synthetic DNA fragments described inSEQ ID NO: 21 and SEQ ID NO: 22 were used as forward and reverseprimers, respectively.

[0228] Each template cDNA (3 μl) was mixed with 1033 Vogelstein buffer(2.5 μl), 2.5 mM dNTP (1.5 μl), forward primer (1 μl, 25 pmol/μl),reverse primer (1 μl, 25 pmol/μl), β-actin primer mix (each 25 pmol/μl),and EX Taq DNA polymerase (0.2 μl), adjusting the total volume to 25 μl.RT-PCR was carried out with a step of 94° C. for 2 min; 35 cycles of 94°C. for 3 sec, 55° C. for 30 sec, 72° C. for 1 min; and a step of 72° C.for 3 min, and then the samples were kept at 4° C.

[0229] The obtained PCR products were separated by electrophoresis. Theresults were shown in FIGS. 1 (BA2303) and 2 (BA0306).

[0230] It was confirmed that the expression of BA2303 was increased fromday 1 after the vascular endothelium was detached by PTCA, reached themaximal level from about day 2 to day 4, and continued until about day7. The expression of BA0306 was detected over a period from day 1 to day7 after PTCA, with peak expression at day 4.

EXAMPLE 5 Isolation of Long Strand cDNA

[0231] To isolate long strand cDNAs containing the two cDNA fragments(BA2303 and BA0306) obtained in Example 4, RACE (rapid amplificationends)-PCR was performed (Proc. Natl. Acad. Sci. USA (1988) Vol. 85,8998-9002; “PCR Method for Gene Amplification/Principles and NovelApplications” Kyoritsu-Syuppan (1992)).

[0232] The PCR was performed twice using the Marathon cDNA AmplificationKit (CLONTECH) and the cDNA fragments obtained in Example 4 as atemplate.

[0233] BA2303 was amplified by PCR using synthetic DNA primers 5described in SEQ ID NO: 15 and SEQ ID NO: 19 (1), and with primersdescribed in SEQ ID NO: 16 and SEQ ID NO: 20 (2), and subsequently usingsynthetic DNA primers described in SEQ ID NO: 17 and SEQ ID NO: 19 (3),and with primers described in SEQ ID NO: 18 and SEQ ID NO: 20 (4).

[0234] BA0306 was amplified by PCR using synthetic DNA primers describedin SEQ ID NO: 23 and SEQ ID NO: 19 (1), and with primers described inSEQ ID NO: 24 and SEQ ID NO: 20 (2), and subsequently using syntheticDNA primers described in SEQ ID NO: 25 and SEQ ID NO: 19 (3), and withprimers described in SEQ ID NO: 26 and SEQ ID NO: 20 (4). The above PCRproduced DNAs described in SEQ ID NO: 1 (BA2303) and in SEQ ID NO: 7(BA0306).

[0235] Analysis of the deduced amino acid sequence by plotting thehydrophilicity and hydrophobicity and by PSORT program suggested thatBA2303 is a protein having seven transmembrane regions (FIG. 3).

EXAMPLE 6 Isolation of Human Counterpart Genes

[0236] The rabbit cDNAs (BA2303 and BA0306) obtained in Example 5 wereused as a probe to screen a human cDNA library (Fetal Brain, STRATAGENE,code:937-227) by colony hybridization according to the standard method(“Gene Engineering HandBook” Jikken-Igaku Zokan, Yodosya, (1992)). Thus,human homologues containing the nucleotide sequences described in SEQ IDNO: 3 (BA2303) and in SEQ ID NO: 9 (BA0306) were obtained.

[0237] Analysis of the deduced amino acid sequence of BA0306 protein byplotting the hydrophilicity and hydrophobicity and by PSORT programsuggested that the protein has 10 transmembrane regions (FIG. 4). It isalso suggested that human BA2303 is a protein having seven transmembraneregions as is the rabbit one obtained in Example 5 (FIG. 5).

[0238] Using the respective human DNA as a probe, the expression of mRNAof the two genes in various human tissues was examined using the HumanMultiple Tissue Northern Blot (CLONTECH, code: #7760-1, #7759-1).

[0239] BA2303 mRNA was expressed in various human tissues as evident astwo bands of about 3.9 kb and about 2.1 kb (FIG. 6).

[0240] BA0306 mRNA was also expressed in various human tissues asdetected as two bands of about 3.5 kb and about 4.4 kb (FIG. 7).

[0241] Homology search between known proteins indicated that humanBA0306 has sequence homology at the amino acid level with S. cerevisiaeoxidative stress resistance protein, S. cerevisiae zinc/cadmiumresistance protein, and heavy metal ion resistance protein, etc.

EXAMPLE 7 Isolation of Mouse BA2303 cDNA

[0242] As was described in Example 6, rabbit BA2303 gene was used as aprobe for screening a mouse cDNA library (STRATAGENE, code: 936-309),and the mouse homologue containing the nucleotide sequence described inSEQ ID NO: 5 was isolated. The deduced amino acid sequence of the codingregion was described in SEQ ID NO: 6.

[0243] Analysis of the deduced amino acid sequence by plotting thehydrophilicity and hydrophobicity and by PSORT program suggested thatmouse BA2303 protein has seven transmembrane regions as do rabbit andhuman BA2303 (FIG. 8).

[0244] BA2303 proteins of the present invention, from rabbit, human andmouse, have a high sequence homology at the amino acid level betweeneach other (FIG. 9). EXAMPLE 8

Isolation of Mouse BA0306 cDNA

[0245] As was described in Example 6, rabbit BA0306 gene was used as aprobe for screening a mouse cDNA library (STRATAGENE, code: 936-309),and the mouse homologue containing the nucleotide sequence described inSEQ ID NO: 27 was isolated. The deduced amino acid sequence of thecoding region was described in SEQ ID NO: 28.

[0246] BA0306 proteins of the present invention, from rabbit, human andmouse, have a high sequence homology at the amino acid level with eachother (FIG. 10).

EXAMPLE 9 Preparation of Anti-peptide Antibody Against Human BA2303

[0247] An oligopeptide(Gln-Asp-Ala-Gln-Gly-Gln-Arg-Ile-Gly-His-Phe-Glu-Phe-His-Gly) containingamino acid residues from 35 to 49 in the sequence described in SEQ IDNO: 4 was synthesized. Two rabbits were immunized three times withpeptide and Freund's complete adjuvant. The rabbit sera obtained aftereach immunization were subjected to ELISA using horse radishperoxidase-conjugated goat anti-rabbit IgG and microplates having wellscoated with the peptide (1 μg/well), and the fluorescence intensity wasmeasured at 492 nm to determine the antibody titers. Titers weredetermined as dilution of sera to obtain a fluorescence intensity at 492nm not more than 0.2. The result showed that the titers of antiserataken from a rabbit A were 50-fold or less before immunization (3 to 5ml), 30,600-fold after the first immunization (16 ml), 40,900-fold afterthe second immunization (25 ml), and 41,100-fold after the thirdimmunization (23 ml), indicating that the titer was increased with thenumber of immunization. The titers of antisera from the other rabbit Bwere not more than 50-fold before immunization (3 to 5 ml), 149,200-fold after the first immunization (25 ml), 327,500-fold after the secondimmunization (25 ml), and 500,000-fold or more after the thirdimmunization (25 ml), indicating that the titer was increased and thatantibody against the peptide was produced.

[0248] Next, the forth immunization was performed on both rabbits A andB. The titers after the forth immunization were 46,500-fold in rabbit A,and 500,000-fold or more in rabbit B as was after the thirdimmunization. Then, the antisera taken from rabbit A after the forthimmunization were purified by affinity chromatography using a columnabsorbed with the peptide that had been used as an antigen. The titer ofthe sera from rabbit A after purification was 69,800-fold.

EXAMPLE 10 Preparation of Recombinant Fusion Protein with Human BA2303Protein

[0249] Fusion proteins of the present invention were prepared as afusion protein with maltose binding protein (MBP) using the expressionplasmid pMAL-C2 (New England Bio Labs. (NEB)), which contains a DNAencoding MBP. Experimental procedures were performed according to themanufacturer's instructions (Catalogue number: #800, ‘Protein Fusion &Purification System’ Ver. 3.03, 12/1994 revised) and by the standardmethod of recombinant DNA technology.

[0250] Using a template of the DNA encoding human BA2303 (SEQ ID NO: 3),which was cloned in the previous Example, a DNA containing thenucleotide sequence corresponding to the N-terminal amino acids(residues 22 (Gly) to 171 (His)), having EcoRI and HindIII restrictionsites at 5′ and 3′ termini, respectively, was amplified by PCR accordingto the standard method. Oligonucleotides described in SEQ ID NO: 29 andSEQ ID NO: 30 were used as 5′ and 3′ primers, respectively. The abovepMAL-C2 expression plasmid (NEB, inserted with a DNA encoding MBP) wasdigested with EcoRI and HindIII, and the resulting fragments wererecovered. Using a commercially available DNA ligation kit, the abovePCR products of human BA2303 were ligated into the pMAL-C2, and theresulting plasmid was used to transfected E. coli TB1 cells. Thebacterial expression plasmid was prepared in a large quantity from thetransformed colony. A culture of the transformed colony ({fraction(1/100)} volume) was inoculated into 1 liter of LB broth containingampicillin and glucose, and incubated with shaking until the OD valuebecame up to 0.5. Then, isopropanol-β-D-thiogalactopyranoside (IPTG) wasadded to the culture to the final concentration of 0.3 mM, and shakingculture was performed further (3 hr). The culture was then centrifugedto remove the supernatant, and the precipitated bacteria was resuspendedin cold column buffer (50 ml, composition: 20 mM Tris-HCl, 200 mM NaCl,1 mM EDTA, and 10 mM mercaptoethanol), which was supplemented with 0.1 MPMSF (50 μl, phenylmethylsulfonyl fluoride) to suppress proteasedigestion.

[0251] The following procedures were carried out on ice unless otherwisenoted. The obtained bacteria suspension was sonicated on ice to disruptcells. Then, the suspension was centrifuged (9000 rpm, 15 to 30 min) torecover soluble fraction. The soluble fraction was diluted with ice-coldcolumn buffer to load on a column.

[0252] Amylose resin (15 ml, BIORAD) was packed in a disposable column(2.5 dia.×10 cm), washed, and equilibrated with 8 volumes of ice-coldcolumn buffer. The sample was loaded onto the column using a pump tokeep the flow rate 1 ml/min, and washed with ice-cold column buffer.

[0253] The fusion protein was eluted and fractionated with ice-coldcolumn buffer containing 10 mM maltose. Each fraction was separated bySDS-PAGE, and analyzed by western blotting using antisera against MBP(NEB). Fractions producing a band detected by western blotting at theposition approximately corresponding to that of the full-length fusionprotein were determined to be positive. Next, the positive fractionswere further purified. MBP/BA2303 fusion protein can be digested byadding 1 mg/ml factor Xa (5 μl) to the solution containing the fusionprotein and incubating it for 24 hr. Digestion of the fusion protein canbe determined by SDS-PAGE followed by western blotting using antiseraagainst MBP.

EXAMPLE 11 Preparation of Antibody Against Human BA2303 Protein

[0254] Recombinant protein prepared in Example 10, containingapproximately 150 N-terminal amino acids of human BA2303 protein(residues 22 (Gly) to 171 (His)), was used as an immunogen. Two rabbitswere immunized with the recombinant protein and Freund's completeadjuvant. The rabbit sera was subjected to ELISA using horse radishperoxidase-conjugated goat anti-rabbit IgG and microplates having wellscoated with the peptide (1 μg/well), and the fluorescence intensity wasmeasured at 492 nm to determine the antibody titers. The titer wasdetermined as dilution of serum to obtain a fluorescence intensity at492 nm not more than 0.2. The result showed that the titer of sera takenfrom a rabbit was 50-fold or less before immunization (3to 5 ml), and316,900- fold after immunization (18 ml), indicating that the titer wasincreased. The titer of the sera from the other rabbit was less than50-fold before immunization (3 to 5 ml), and increased to 312,300-foldafter immunization (23 ml), indicating that antibody against therecombinant protein was produced.

EXAMPLE 12 Construction of an Expression Vector for Recombinant HumanBA2303

[0255] Using a template of the DNA encoding human BA2303 (SEQ ID NO: 3),which was cloned in the previous Example, a DNA containing thenucleotide residues 77 to 1419 (containing the entire open reading frame(ORF)), having XbaI restriction sites at both 5′ and 3′ termini, wasamplified by PCR according to the standard method. Oligonucleotidesdescribed in SEQ ID NO: 31 and in SEQ ID NO: 32 were used as 5′ and 3′primers, respectively.

[0256] The resulting PCR products were ligated into the XbaI site of thepcDNA expression plasmid (Invitrogen) using a commercially available DNAligation kit to construct an expression vector for recombinant humanBA2303. Higher eukaryotic host cells such as COS cells can betransfected with the vector, and the resulting colonies are selected toobtain transfected cells. Human BA2303 proteins can be expressedabundantly on the cell surface of the transfected cells by incubatingthe them in appropriate medium such as DMEM containing 10% FCS.

EXAMPLE 13 Preparation of Recombinant Rabbit BA0306 Protein

[0257] Using a template of the DNA encoding rabbit BA0306 (SEQ ID NO:7), which was cloned in the previous Example, a DNA containing thenucleotide residues 2017 (Ile) to 2196 (Met), having BamHI and SalIrestriction sites at 5′ and 3′ termini, respectively, was amplified byPCR according to the standard method. In the amino acid sequence (60residues) encoded by the rabbit nucleotide sequence (nucleotides 2017(Ile) to 2196 (Met)), 58 residues are the same as those in thecorresponding human BA0306 sequence (residues 535 to 594 in SEQ ID NO:10). Oligonucleotides described in SEQ ID NO: 33 and in SEQ ID NO: 34were used as 5′ and 3′ primers, respectively.

[0258] The expression plasmid pQE-32 (QIA expression type IV construct,QIAGEN) was digested with BamHI and SalI, and then blunted.

[0259] According to the instruction manual for handling pQE-32, theobtained PCR products were ligated into the blunted ends of pQE-32digested with BamHI-SalI using a commercially available DNA ligationkit. The resulting expression vector for recombinant human BA0306 wasnamed as pQE-32R7-15.

[0260] Next, E. coli cells (XL-1 blue MRF') were transformed with thepQE-32R7-15 according to the standard method, and the transformedcolonies were selected (“Gene Engineering Handbook” Jikken-IgakuBessatsu, Yodosha (1992) 46-51). A culture of the transformed cells wasinoculated into LB broth containing ampicillin and glucose, andincubated at 37° C. with shaking, with measuring the OD. Then, IPTG(isopropanol-β-D-thiogalactopyranoside) was added to the culture to thefinal concentration of 1 mM, and shaking culturing was further performedat 37° C. for 4 hrs. The culture was centrifuged to remove thesupernatant, and the precipitated bacteria was resuspended in columnbuffer. The suspension was sonicated on ice to disrupt cells, thencentrifuged, and soluble fraction was recovered. The soluble fractionwas diluted with ice-cold column buffer to load on a column.

[0261] A column was packed with Ni-NTA resin, washed, and equilibratedwith column buffer. The samples were applied on the column and washedwith column buffer. The eluted fractions were collected, and thusrecombinant rabbit BA0306 protein was obtained.

EXAMPLE 14 Preparation of Antibody Against Human BA0306

[0262] Recombinant rabbit BA0306 protein prepared in Example 13 was usedas an immunogen. The protein and Freund's complete adjuvant were used toimmunize chickens. The chicken sera were subjected to ELISA using horseradish peroxidase-conjugated anti-chicken IgG and microplates havingwells coated with the recombinant protein (1 μg/well), and thefluorescence intensity was measured to determine the antibody titers.The result showed that the titer was increased, indicating that antibodyagainst the ecombinant protein was produced.

[0263] Furthermore, rabbit BA0306 protein fragment, which was used as animmunogen in this example, and the above recombinant human BA0306protein were detected by western blotting using the chicken antisera,indicating that the antisera had a cross reactivity with human BA0306protein.

EXAMPLE 15 Generation of Knockout Mice of Mouse BA2303 gene

[0264] A knockout mouse, whose endogenous gene encoding mouse BA2303protein was inactivated, was generated as follows.

[0265] (1) Construction of a Targeting Vector

[0266] A targeting vector for generation of a knockout mouse, in whichan endogenous gene encoding mouse BA2303 protein was inactivated(knocked out) by homologous recombination (Nikkei-Science (1994) May,52-62), was constructed as follows.

[0267] The cDNA encoding mouse BA2303 protein (SEQ ID NO: 5), which wascloned in the previous Example, was labeled with ³²P by the standardmethod to obtain a probe used in hybridization. The probe was used toscreen a cosmid mouse genomic DNA library (“Labomanual Human GenomeMapping” Hori M., and Nakamura Y. edit., Maruzen Syuppan), and thus, amouse genomic DNA clone containing exons (E1, E2, and E3) which encodemouse BA2303 protein was isolated. The structure of the genomic DNA wasschematically shown in FIG. 11. The genomic DNA was subcloned into aplasmid, and digested with SacII to remove the region of 124 bpencompassing E1 and the intron between E1 and E2, and then ligated withan insert of a neomycin resistance gene of 1143bp (neo, as a positiveselection marker), which had been digested with restriction enzymes andblunted.

[0268] The plasmid pBluescript II SK(−) was digested with SacII, andligated with an insert of a thymidine kinase gene (TK, as a negativeselection marker). Then, the resulting pBluescript II SK(−) was digestedwith XbaI, and ligated with an insert of the above mouse BA2303 genomicDNA having a neo gene insertion.

[0269] (2) Transfection of the Targeting Vector into ES Cells

[0270] Mouse embryonic stem cells (ES cells) (Nature (1993) 362,255-258; Nature (1987) 326, 292-295), which were cultured in DMEMcontaining 15% fetal bovine serum, were trypsinized to obtain singleisolated cells, washed three times in phosphate buffer, and prepared asa cell suspension of 1×10⁷ cells/ml. The targeting vector was added tothe cells (25 μg/1 ml cell suspension), and electroporation wasperformed with a single pulse of 350 V/cm (25 μF). Then, the ES cellswere seeded into 10 cm dishes (1×107 cells/dish), cultured for one dayin maintenance medium, and then the medium was replaced with selectionmedium (containing G418 (250 μg/ml) and 2 μM gancyclovir). The culturewas continued with replacing the medium every two days. On the tenth dayafter transfection of targeting vector, 540 neomycin resistant ES cloneswere isolated using a micropipet under microscopic observation. Theclones were cultured separately in 24 well plates layered with feedercells, and replica of 540 neomycin resistant ES cells were obtained.

[0271] (3) Screening of Knockout ES Cells

[0272] Each neomycin resistant ES clone was examined by PCR whether itsendogenous gene encoding mouse BA2303 protein was inactivated (knockedout) by homologous recombination.

[0273] PCR was performed using genomic DNA extracted from each neomycinresistant ES clone as a template, with two primers designed based on thesequence of the neo gene (SEQ ID NO: 36 and SEQ ID NO: 37) (1) and onthe mouse BA2303 genomic DNA sequence which locates on the flankingregion of the BA2303 DNA which was inserted in the targeting vector (SEQID NO: 35 and SEQ ID NO: 38) (2). DNA was purified using an automatedDNA purification robot (Kubota). The result showed that desired PCRproducts were obtained in several clones among the ES clones examined.Further selection of these clones can be performed by genomic Southernblotting. Genomic DNA was extracted from each clone, digested withrestriction enzymes, and separated by electrophoresis on an agarose gel.Then, the DNA was transferred onto a nylon membrane, and subjected tohybridization using a probe designed based on the genomic sequence ofmouse BA2303. The probe was designed based on the sequence which locatesin the flanking region of the site of homologous recombination, and thusenabled to distinguish mutated genome from normal one by size. Theknockout ES clone selected in this way was used for generation ofknockout mice as described below.

[0274] (4) Generation of Knock Out Mice

[0275] The above obtained ES cells, having inactivation in theendogenous gene encoding mouse BA2303 protein as a result of homologousrecombination, were injected into blastocysts obtained by crossingC57BL6 mice (Japan Charles River) (15 cells/embryo, microinjection).Immediately after microinjection, the blastocysts were implanted intouterines of ICR mice (Clea Japan), which had undergone pseudopregnancytreatment two days and half before (10 blastocysts/one side of theuterine). Thus, desired chimera mice were obtained. The chimera werecrossed with normal C57BL6 mice to obtain agouti mice, whose color isattributed to a gene determining hair color, originating from ES cells.

EXAMPLE 16 Generation of Knockout Mice of Mouse BA0306 Gene

[0276] (1) Construction of a Targeting Vector.

[0277] A targeting vector for generation of a knockout mouse, in whichthe endogenous gene encoding mouse BA0306 protein was inactivated(knocked out) by homologous recombination (Nikkei-Science (1994) May,52-62), was constructed as follows.

[0278] The cDNA encoding mouse BA0306 protein (SEQ ID NO: 27), which wascloned in the previous Example, was labeled with ³²p by the standardmethod to obtain a probe used in hybridization. The probe was used toscreen a 129SVJ mouse genomic DNA library (STRATAGENE), and a mousegenomic DNA clone containing exons (exon I, II, III, IV, and V) thatencode mouse BA0306 protein was isolated.

[0279] The plasmid pBluescript II SK(−) was digested with XhoI andHindIII, and ligated with XhoI-HindIII-digested thymidine kinase gene(TK, as a negative selection marker). Next, NotI-digested pBluescript IISK(−) was ligated with an insert of the above mouse BA0306 genomic DNA(exons I to V). Then, the neomycin resistance gene (neo, as a positiveselection marker) was digested with BamHI and XhoI, blunted, and ligatedinto the Aor51HI site of the exon V in the mouse BA0306 genomic DNA.Finally, the resulting pBluescript II SK(−) was digested with SacII andlinealized to use as a targeting vector.

[0280] (2) Transfection of the Targeting Vector into ES Cells.

[0281] Mouse embryonic stem cells (ES cells, 1×10⁸ cells) (Nature (1993)362, 255-258; Nature (1987) 326, 292-295), which were cultured in DMEMcontaining 15% fetal bovine serum, were trypsinized to obtain singleisolated cells, washed three times in phosphate buffer, and thenprepared as a cell suspension of 1×10⁷ cells/ml. The targeting vectorwas added to the (25 μg/l ml cell suspension), and electroporation wasperformed with a single pulse of 350 V/cm (25 μF). Then, the ES cellswere seeded into 10 cm dishes (1×10⁷ cells/dish), cultured 1 day inmaintenance medium, and then the medium was replaced with selectionmedium (containing G418 (250 μg/ml) and 2 μM gancyclovir). The culturewas continued with replacing the medium every two days. On the tenth dayafter transfection, 573 neomycin resistant ES clones were isolated usinga micropipet under microscopic observation. The clones were culturedseparately in 24 well plates layered with feeder cells, and replica of573 neomycin resistant ES cells were obtained.

[0282] (3) Screening of Knockout ES Cells

[0283] Each neomycin resistant ES clone was examined by genomic Southernblotting whether its endogenous gene encoding mouse BA0306 protein wasinactivated (knocked out) through homologous recombination.

[0284] Genomic DNA was extracted from each neomycin resistant ES clone,and genomic Southern blotting was performed on EcoRI digested genomicDNA fragments according to the standard method using the followingprobes.

probe 1

[0285] 5′ flanking DNA which was amplified using two primers describedin SEQ ID NO: 39 and SEQ ID NO: 40.

probe 2

[0286] 3′ flanking DNA which was amplified using two primers describedin SEQ ID NO: 41 and SEQ ID NO: 42. DNA was purified using an automatedDNA purification robot (Kubota).

[0287] If the endogenous gene encoding BA0306 is normally targeted bythe targeting vector, the 5′ and 3′ flanking genes encompassing theintegrated neo gene can be detected as 7 kb and 5 kb bands,respectively.

[0288] The result showed that desired knockout of the gene was occurredin three ES clones (named as 0-16-9, 0-22-11, and 0-22-18), which wereused for generation of knockout mice as described below.

[0289] (4) Generation of Knock Out Mice

[0290] The ES clones obtained above, having inactivation in theendogenous gene encoding mouse BA0306 protein as a result of homologousrecombination, were microinjected into blastocysts obtained by crossingC57BL6 mice (Japan Charles River) (15 cells/embryo). Immediately aftermicroinjection, the blastocysts were transferred to uterines of ICR mice(Clea Japan) (10 blastocysts/one side of the uterine), which hadundergone pseudopregnancy treatment two days and half before. As aresult, desired knockout chimera mice were obtained from each ES cloneas followings.

[0291] (clone 0-16-9)

[0292] Total number of injected cells: 83

[0293] Littermates: 13

[0294] Chimera mice: 7

[0295] Chimera where contribution to hair color is 80% or more:

[0296] 2

[0297] (clone 0-22-11)

[0298] Total number of injected cells: 202

[0299] Littermates: 12

[0300] Chimera mice: 3

[0301] Chimera where contribution to hair color is 80% or more:

[0302] 3

[0303] (clone 0-22-18)

[0304] Total number of injected cells: 148

[0305] Littermates: 9

[0306] Chimera mice: 5

[0307] The chimera were crossed with normal C57BL6 mice to obtain agoutimice whose color is attributed to a gene determining hair color,originating from ES cells.

Industrial Applicability

[0308] The present invention provides two novel physiologically activeprotein molecules (BA0306, and BA2303) having characteristics describedbelow, which are specifically expressed in arteriosclerosis or coronaryrestenosis, and are predicted to relate closely to the onset andprogress of these diseases; their fragments; a gene (DNA) encoding theprotein molecules; an antibody reactive with the molecule, and itsfragment; and pharmaceutical compositions comprising the above proteinmolecule or the antibody.

[0309] [BA0306]

[0310] A molecule having the following characteristics, and presumed tohave inhibitory effects on active oxygen species such as nitrogenmonoxide (NO), which has been identified to be involved in the progressof arteriosclerosis and restenosis.

[0311] (1) Its expression is increased from day 1 to day 7 after PTCA ofthe coronary aorta (peak at day 4).

[0312] (2) Its mRNA is expressed in various human tissues as detected byNorthern blotting as approximately 3.5 kb and 4.4 kb bands.

[0313] (3) Its 10 predicted transmembrane regions.

[0314] (4) Its sequence homology at the amino acid level with S.cerevisiae oxidative stress resistance protein, S. cerevisiaezinc/cadmium resistance protein, and heavy metal ion resistance protein,etc.

[0315] [BA2303]

[0316] A molecule having the following characteristics, and presumed tobe a G protein(GTP binding protein)-coupled receptor that transduces aspecific signal through intracellular G protein to an effector on theplasma membrane or in the cytoplasm by binding to an in vivo ligandwhich is involved in the onset and progress of arteriosclerosis andrestenosis.

[0317] (1) Its expression is increased day 1 after PTCA of the coronaryaorta, reaches the maximum on day 2 to day 4, and continued until day 7.

[0318] (2) Its mRNA is expressed in various human tissues as detected byNorthern blotting as approximately 3.9 kb and 2.1 kb bands.

[0319] (3) having seven predicted transmembrane regions.

[0320] Therefore, a gene (DNA) or protein of the present invention orits part, and an antibody reactive with the protein, or a part of theantibody are extremely useful in developing the drugs targeting the geneor the protein molecule for treatment and prevention of arteriosclerosisas well as restenosis after PTCA of arterial embolism. Also, the DNAitself is very useful as an antisense medicine, the extracellular domainfragment of the protein is useful as a soluble receptor medicine, andthe antibody or its part is useful as an antibody medicine.

[0321] Furthermore, the gene (DNA), protein, and antibody of the presentinvention are useful as a reagent for screening a protein (ligand)interacting with the protein of the invention, identification of thefunction of the ligand, and developing a drug which targets the ligand.

[0322] In addition, based on the nucleotide sequence originating fromrabbit or mouse, as an embodiment of the DNA of the present invention,model animals (knockout animals) can be generated by disrupting(inactivating) a corresponding endogenous gene. Similarly, trasngenicanimals can be generated as a model animal by introducing human DNA, asan embodiment of the DNA of the present invention, into mammals such asmice except human. It is possible to identify the functions of the geneand protein of the invention by analyzing the physical, biological,pathological, and genetical characteristics of the model animals.

[0323] Moreover, it is possible to generate model animals having a humangene of the invention alone by crossing the model animals, whoseendogenous gene is disrupted, with the transgenic animals. Thus, it ispossible to estimate the therapeutic effects of a drug which targets theintroduced human gene (compounds, and antibodies, etc.) byadministrating the drug into the model animals.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 42 <210> SEQ ID NO 1<211> LENGTH: 1399 <212> TYPE: DNA <213> ORGANISM: Oryctolagus cuniculus<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1158) <400>SEQUENCE: 1 gtc aga atc aac aac ata gca gta gct gta gga aaa gaa gct aaactt 48 Val Arg Ile Asn Asn Ile Ala Val Ala Val Gly Lys Glu Ala Lys Leu 15 10 15 tac ctg ttc caa gcc cag gaa tgg ctg aag ctg cag gaa agc agt cat96 Tyr Leu Phe Gln Ala Gln Glu Trp Leu Lys Leu Gln Glu Ser Ser His 20 2530 gat tac agc tgt cat gaa aaa tta tcc aaa gcc caa ttg aca atg acc 144Asp Tyr Ser Cys His Glu Lys Leu Ser Lys Ala Gln Leu Thr Met Thr 35 40 45atg aac cag agt gaa cat aat atg aca gtg tcc cag att cca tct cca 192 MetAsn Gln Ser Glu His Asn Met Thr Val Ser Gln Ile Pro Ser Pro 50 55 60 caaacg tgg cac gtg ttt tat gca gac aag tat aca tgc cga gtt gac 240 Gln ThrTrp His Val Phe Tyr Ala Asp Lys Tyr Thr Cys Arg Val Asp 65 70 75 80 gaggag aat tgg caa gtg gaa gat atc cca ttt gaa atg gtg tta cta 288 Glu GluAsn Trp Gln Val Glu Asp Ile Pro Phe Glu Met Val Leu Leu 85 90 95 aac ccagat gct gaa gga aat ccg ttt gat cat ttt ggt gct gga gaa 336 Asn Pro AspAla Glu Gly Asn Pro Phe Asp His Phe Gly Ala Gly Glu 100 105 110 tct gggtta cat gag ttc ttt ttc ctc cta gtc cta gtg tac ttt gtg 384 Ser Gly LeuHis Glu Phe Phe Phe Leu Leu Val Leu Val Tyr Phe Val 115 120 125 act gcttgc att tat gcg cag tca ttg tgg cag gct ctt aag aaa gga 432 Thr Ala CysIle Tyr Ala Gln Ser Leu Trp Gln Ala Leu Lys Lys Gly 130 135 140 ggg cccatg cac atg att cta aag gtg ctg aca act gca ctg ctg ttg 480 Gly Pro MetHis Met Ile Leu Lys Val Leu Thr Thr Ala Leu Leu Leu 145 150 155 160 caagct ggt tca gct gta gct aat tac atc cat ttc tcc agt tac tcc 528 Gln AlaGly Ser Ala Val Ala Asn Tyr Ile His Phe Ser Ser Tyr Ser 165 170 175 aaagat gga atc ggg gta cct ttt atg gga agc ttg gca gaa ttt ttt 576 Lys AspGly Ile Gly Val Pro Phe Met Gly Ser Leu Ala Glu Phe Phe 180 185 190 gacatc gct tcc caa att cag atg tta tac ctg ctt ctg agt ctg tgc 624 Asp IleAla Ser Gln Ile Gln Met Leu Tyr Leu Leu Leu Ser Leu Cys 195 200 205 atgggc tgg acc ata gtc agg atg aag aag tct caa agc aga cct ctc 672 Met GlyTrp Thr Ile Val Arg Met Lys Lys Ser Gln Ser Arg Pro Leu 210 215 220 cagtgg gat tcg acc cct gcc tcc act ggc att gcc gtg ttc att gtc 720 Gln TrpAsp Ser Thr Pro Ala Ser Thr Gly Ile Ala Val Phe Ile Val 225 230 235 240ctg aca cag agt gtt ttg ctg ctt tgg gaa cag ttt gaa gat acc ggt 768 LeuThr Gln Ser Val Leu Leu Leu Trp Glu Gln Phe Glu Asp Thr Gly 245 250 255cat cat agc tcc cat tca cac cac aac tta gca ggg atc ctt ctg atc 816 HisHis Ser Ser His Ser His His Asn Leu Ala Gly Ile Leu Leu Ile 260 265 270gtt tta aga att tgc ctg gca ttg tca tta ggc tgt gga ctc tat cag 864 ValLeu Arg Ile Cys Leu Ala Leu Ser Leu Gly Cys Gly Leu Tyr Gln 275 280 285atc atc aca gtg gag agg agc aca ctc aaa agg gag ttc tac atc aca 912 IleIle Thr Val Glu Arg Ser Thr Leu Lys Arg Glu Phe Tyr Ile Thr 290 295 300ttt gcc aaa ggc tgt atc tta tgg ttt ttg tgc cat cca agt ctg gca 960 PheAla Lys Gly Cys Ile Leu Trp Phe Leu Cys His Pro Ser Leu Ala 305 310 315320 tgc att tct gtc att ttt aat gac tac caa aga gat aag gtt att aca 1008Cys Ile Ser Val Ile Phe Asn Asp Tyr Gln Arg Asp Lys Val Ile Thr 325 330335 ata ggt gtt atc ctt ggc cag tct gtt gcc atg gtt atc ctc tac aga 1056Ile Gly Val Ile Leu Gly Gln Ser Val Ala Met Val Ile Leu Tyr Arg 340 345350 ctc ttt ctc tcc cac agt cta tac tgg gaa gtt tct tcc ctt tcc tca 1104Leu Phe Leu Ser His Ser Leu Tyr Trp Glu Val Ser Ser Leu Ser Ser 355 360365 gta aca cta cca ctg acc gta tcg tct gga cac aaa agc cgc cct cat 1152Val Thr Leu Pro Leu Thr Val Ser Ser Gly His Lys Ser Arg Pro His 370 375380 ttc tga tacttgattt ctgtggaaaa gaaaagtgaa ggggttaaaa gagtgcaata 1208Phe 385 aggacccaaa tacagtgact tttttttcat acatttggta tgaaaaatcgaatagcaaaa 1268 gcagagcatg tttctgtgat aactgcattt aagcagtacc aaaactgaacaaaggtaata 1328 actgaaatgt tttaaaatac atgtaaacaa taaactttca ggaaattctgttgttaaaaa 1388 aaaaaaaaaa c 1399 <210> SEQ ID NO 2 <211> LENGTH: 385<212> TYPE: PRT <213> ORGANISM: Oryctolagus cuniculus <400> SEQUENCE: 2Val Arg Ile Asn Asn Ile Ala Val Ala Val Gly Lys Glu Ala Lys Leu 1 5 1015 Tyr Leu Phe Gln Ala Gln Glu Trp Leu Lys Leu Gln Glu Ser Ser His 20 2530 Asp Tyr Ser Cys His Glu Lys Leu Ser Lys Ala Gln Leu Thr Met Thr 35 4045 Met Asn Gln Ser Glu His Asn Met Thr Val Ser Gln Ile Pro Ser Pro 50 5560 Gln Thr Trp His Val Phe Tyr Ala Asp Lys Tyr Thr Cys Arg Val Asp 65 7075 80 Glu Glu Asn Trp Gln Val Glu Asp Ile Pro Phe Glu Met Val Leu Leu 8590 95 Asn Pro Asp Ala Glu Gly Asn Pro Phe Asp His Phe Gly Ala Gly Glu100 105 110 Ser Gly Leu His Glu Phe Phe Phe Leu Leu Val Leu Val Tyr PheVal 115 120 125 Thr Ala Cys Ile Tyr Ala Gln Ser Leu Trp Gln Ala Leu LysLys Gly 130 135 140 Gly Pro Met His Met Ile Leu Lys Val Leu Thr Thr AlaLeu Leu Leu 145 150 155 160 Gln Ala Gly Ser Ala Val Ala Asn Tyr Ile HisPhe Ser Ser Tyr Ser 165 170 175 Lys Asp Gly Ile Gly Val Pro Phe Met GlySer Leu Ala Glu Phe Phe 180 185 190 Asp Ile Ala Ser Gln Ile Gln Met LeuTyr Leu Leu Leu Ser Leu Cys 195 200 205 Met Gly Trp Thr Ile Val Arg MetLys Lys Ser Gln Ser Arg Pro Leu 210 215 220 Gln Trp Asp Ser Thr Pro AlaSer Thr Gly Ile Ala Val Phe Ile Val 225 230 235 240 Leu Thr Gln Ser ValLeu Leu Leu Trp Glu Gln Phe Glu Asp Thr Gly 245 250 255 His His Ser SerHis Ser His His Asn Leu Ala Gly Ile Leu Leu Ile 260 265 270 Val Leu ArgIle Cys Leu Ala Leu Ser Leu Gly Cys Gly Leu Tyr Gln 275 280 285 Ile IleThr Val Glu Arg Ser Thr Leu Lys Arg Glu Phe Tyr Ile Thr 290 295 300 PheAla Lys Gly Cys Ile Leu Trp Phe Leu Cys His Pro Ser Leu Ala 305 310 315320 Cys Ile Ser Val Ile Phe Asn Asp Tyr Gln Arg Asp Lys Val Ile Thr 325330 335 Ile Gly Val Ile Leu Gly Gln Ser Val Ala Met Val Ile Leu Tyr Arg340 345 350 Leu Phe Leu Ser His Ser Leu Tyr Trp Glu Val Ser Ser Leu SerSer 355 360 365 Val Thr Leu Pro Leu Thr Val Ser Ser Gly His Lys Ser ArgPro His 370 375 380 Phe 385 <210> SEQ ID NO 3 <211> LENGTH: 2130 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:CDS <222> LOCATION: (97)..(1419) <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: 2001, 2003, 2019, 2033, 2035, 2053, 2079,2114, 2116, 2119, 2129 <223> OTHER INFORMATION: n is a or g or c or t<400> SEQUENCE: 3 ctccggcgcc caccccgcct cccccagctg ccgacgtggg gcgggcagccgccggcggct 60 gggagccgag gcgtcggtgc agacctggag acgggc atg ggg ggg ctgcgg ctg 114 Met Gly Gly Leu Arg Leu 1 5 ctg gct gtg gcc ctc acg tgc tgctgg tgg ccg cag ggc agc cag ggt 162 Leu Ala Val Ala Leu Thr Cys Cys TrpTrp Pro Gln Gly Ser Gln Gly 10 15 20 aag acc ctg cgg ggc agc ttc agc agcacc gcg gcc cag gac gcc cag 210 Lys Thr Leu Arg Gly Ser Phe Ser Ser ThrAla Ala Gln Asp Ala Gln 25 30 35 ggc cag cgc atc ggc cac ttc gag ttc catggt gac cat gct ctt ctg 258 Gly Gln Arg Ile Gly His Phe Glu Phe His GlyAsp His Ala Leu Leu 40 45 50 tgt gtc aga atc aac aac ata gca gta gct gttgga aaa gaa gct aaa 306 Cys Val Arg Ile Asn Asn Ile Ala Val Ala Val GlyLys Glu Ala Lys 55 60 65 70 ctc tac ctg ttc caa gcc cag gaa tgg cta aagcta cag caa agc agt 354 Leu Tyr Leu Phe Gln Ala Gln Glu Trp Leu Lys LeuGln Gln Ser Ser 75 80 85 cat ggt tat agc tgt agt gaa aaa tta tcc aaa gctcag ttg aca atg 402 His Gly Tyr Ser Cys Ser Glu Lys Leu Ser Lys Ala GlnLeu Thr Met 90 95 100 acc atg aac cag acc gaa cat aat ctg aca gtg tcccag att ccg tct 450 Thr Met Asn Gln Thr Glu His Asn Leu Thr Val Ser GlnIle Pro Ser 105 110 115 cca caa acg tgg cat gtg ttt tat gca gac aag tataca tgc caa gat 498 Pro Gln Thr Trp His Val Phe Tyr Ala Asp Lys Tyr ThrCys Gln Asp 120 125 130 gac aag gag aat tct cag gtg gaa gat atc cca tttgaa atg gtg tta 546 Asp Lys Glu Asn Ser Gln Val Glu Asp Ile Pro Phe GluMet Val Leu 135 140 145 150 cta aac cca gat gcc gaa ggg aat cca ttt gatcat ttt agt gct gga 594 Leu Asn Pro Asp Ala Glu Gly Asn Pro Phe Asp HisPhe Ser Ala Gly 155 160 165 gaa tct ggg tta cat gag ttc ttt ttc ctc ctagtc cta gtg tac ttt 642 Glu Ser Gly Leu His Glu Phe Phe Phe Leu Leu ValLeu Val Tyr Phe 170 175 180 gtg att gct tgc att tat gct caa tca ttg tggcag gct att aag aaa 690 Val Ile Ala Cys Ile Tyr Ala Gln Ser Leu Trp GlnAla Ile Lys Lys 185 190 195 ggc gga ccc atg cac atg att tta aag gtt ctgaca act gca ttg ctg 738 Gly Gly Pro Met His Met Ile Leu Lys Val Leu ThrThr Ala Leu Leu 200 205 210 tta caa gct ggt tca gct tta gct aat tac attcat ttc tcc agt tac 786 Leu Gln Ala Gly Ser Ala Leu Ala Asn Tyr Ile HisPhe Ser Ser Tyr 215 220 225 230 tcc aaa gat gga ata ggg gta cca ttt atggga agt ttg gca gaa ttt 834 Ser Lys Asp Gly Ile Gly Val Pro Phe Met GlySer Leu Ala Glu Phe 235 240 245 ttt gac atc gct tcc caa att cag atg ttatac tta ctt ttg agt cta 882 Phe Asp Ile Ala Ser Gln Ile Gln Met Leu TyrLeu Leu Leu Ser Leu 250 255 260 tgc atg ggt tgg aca ata gtc aga atg aagaag tct caa agc aga cct 930 Cys Met Gly Trp Thr Ile Val Arg Met Lys LysSer Gln Ser Arg Pro 265 270 275 ctc cag tgg gat tct acg cct gca tcc actggc att gca gta ttc att 978 Leu Gln Trp Asp Ser Thr Pro Ala Ser Thr GlyIle Ala Val Phe Ile 280 285 290 gtc atg aca cag agt gtt ttg cta ctt tgggaa cag ttt gaa gat atc 1026 Val Met Thr Gln Ser Val Leu Leu Leu Trp GluGln Phe Glu Asp Ile 295 300 305 310 agt cat cat agc tac cat tca cac cacaac tta gca ggg atc ctc cta 1074 Ser His His Ser Tyr His Ser His His AsnLeu Ala Gly Ile Leu Leu 315 320 325 att gtt cta aga att tgc cta gca ttgtca tta ggc tgt gga ctc tat 1122 Ile Val Leu Arg Ile Cys Leu Ala Leu SerLeu Gly Cys Gly Leu Tyr 330 335 340 cag atc atc aca gtg gag aga agt acactc aaa agg gag ttc tac atc 1170 Gln Ile Ile Thr Val Glu Arg Ser Thr LeuLys Arg Glu Phe Tyr Ile 345 350 355 aca ttt gcc aaa ggc tgt atc ttg tggttt tta tgc cat cca gtt ctt 1218 Thr Phe Ala Lys Gly Cys Ile Leu Trp PheLeu Cys His Pro Val Leu 360 365 370 gca tgc att tct gtc att ttt agc gactac caa aga gac aag gtt att 1266 Ala Cys Ile Ser Val Ile Phe Ser Asp TyrGln Arg Asp Lys Val Ile 375 380 385 390 aca ata ggt gtt atc ctt tgc cagtct gtt tcc atg gtt att ctc tac 1314 Thr Ile Gly Val Ile Leu Cys Gln SerVal Ser Met Val Ile Leu Tyr 395 400 405 aga ctc ttt ctg tct cac agt ctatac tgg gaa gtt tct tca ctt tct 1362 Arg Leu Phe Leu Ser His Ser Leu TyrTrp Glu Val Ser Ser Leu Ser 410 415 420 tca gta aca cta cca ctg acc atatca tct gga cac aaa agt cgc cct 1410 Ser Val Thr Leu Pro Leu Thr Ile SerSer Gly His Lys Ser Arg Pro 425 430 435 cat ttc tga tacttgatttttgttgagag gaaaagtgaa ttggttaaaa 1459 His Phe 440 gagtgcaata aggatccaaatacagtgact tttttttcat acatttagta tgaaaacttg 1519 aacagcgaaa gcagagcatgttatttatat aactgcattt aagcagtacc aagactgaaa 1579 aaaaaggtaa taaatgaaatgttttgaaat atacttaaac aacaaacttt gaagaaagtg 1639 ttgttataaa attattgaagcgatttctat gtggaaataa atgtgaaaaa taaaactatg 1699 atattttggt aaaatattcaccacttataa tgcctcatct taatagctaa ctcasgttta 1759 atartcttat aaaaagtaatcagttaaatg aatacttgct tataaatatc taaactaatc 1819 cactttatga aatcagtgttatacattgaa ttttaaaact gctgcctttt atgcctttaa 1879 ggaaaatgtt tttccctattttgaatttta aaggaattga aattcctccc ggaaattaat 1939 ataaataggg ttccccgttaaatgaaataa accctggttt aattggtggg gtggaattaa 1999 tncncccaat tttttcccgncccttttttg gggncncatt ttccgggttt taanccttga 2059 ataaaccaaa gggtttttgnaaaaaccctt tttttgaaaa aaaattaaaa ccttnanttn 2119 cctttacccn g 2130 <210>SEQ ID NO 4 <211> LENGTH: 440 <212> TYPE: PRT <213> ORGANISM: Homosapiens <400> SEQUENCE: 4 Met Gly Gly Leu Arg Leu Leu Ala Val Ala LeuThr Cys Cys Trp Trp 1 5 10 15 Pro Gln Gly Ser Gln Gly Lys Thr Leu ArgGly Ser Phe Ser Ser Thr 20 25 30 Ala Ala Gln Asp Ala Gln Gly Gln Arg IleGly His Phe Glu Phe His 35 40 45 Gly Asp His Ala Leu Leu Cys Val Arg IleAsn Asn Ile Ala Val Ala 50 55 60 Val Gly Lys Glu Ala Lys Leu Tyr Leu PheGln Ala Gln Glu Trp Leu 65 70 75 80 Lys Leu Gln Gln Ser Ser His Gly TyrSer Cys Ser Glu Lys Leu Ser 85 90 95 Lys Ala Gln Leu Thr Met Thr Met AsnGln Thr Glu His Asn Leu Thr 100 105 110 Val Ser Gln Ile Pro Ser Pro GlnThr Trp His Val Phe Tyr Ala Asp 115 120 125 Lys Tyr Thr Cys Gln Asp AspLys Glu Asn Ser Gln Val Glu Asp Ile 130 135 140 Pro Phe Glu Met Val LeuLeu Asn Pro Asp Ala Glu Gly Asn Pro Phe 145 150 155 160 Asp His Phe SerAla Gly Glu Ser Gly Leu His Glu Phe Phe Phe Leu 165 170 175 Leu Val LeuVal Tyr Phe Val Ile Ala Cys Ile Tyr Ala Gln Ser Leu 180 185 190 Trp GlnAla Ile Lys Lys Gly Gly Pro Met His Met Ile Leu Lys Val 195 200 205 LeuThr Thr Ala Leu Leu Leu Gln Ala Gly Ser Ala Leu Ala Asn Tyr 210 215 220Ile His Phe Ser Ser Tyr Ser Lys Asp Gly Ile Gly Val Pro Phe Met 225 230235 240 Gly Ser Leu Ala Glu Phe Phe Asp Ile Ala Ser Gln Ile Gln Met Leu245 250 255 Tyr Leu Leu Leu Ser Leu Cys Met Gly Trp Thr Ile Val Arg MetLys 260 265 270 Lys Ser Gln Ser Arg Pro Leu Gln Trp Asp Ser Thr Pro AlaSer Thr 275 280 285 Gly Ile Ala Val Phe Ile Val Met Thr Gln Ser Val LeuLeu Leu Trp 290 295 300 Glu Gln Phe Glu Asp Ile Ser His His Ser Tyr HisSer His His Asn 305 310 315 320 Leu Ala Gly Ile Leu Leu Ile Val Leu ArgIle Cys Leu Ala Leu Ser 325 330 335 Leu Gly Cys Gly Leu Tyr Gln Ile IleThr Val Glu Arg Ser Thr Leu 340 345 350 Lys Arg Glu Phe Tyr Ile Thr PheAla Lys Gly Cys Ile Leu Trp Phe 355 360 365 Leu Cys His Pro Val Leu AlaCys Ile Ser Val Ile Phe Ser Asp Tyr 370 375 380 Gln Arg Asp Lys Val IleThr Ile Gly Val Ile Leu Cys Gln Ser Val 385 390 395 400 Ser Met Val IleLeu Tyr Arg Leu Phe Leu Ser His Ser Leu Tyr Trp 405 410 415 Glu Val SerSer Leu Ser Ser Val Thr Leu Pro Leu Thr Ile Ser Ser 420 425 430 Gly HisLys Ser Arg Pro His Phe 435 440 <210> SEQ ID NO 5 <211> LENGTH: 1918<212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (82)..(1383) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (469)..(471) <223> OTHERINFORMATION: n is a or g or c or t Xaa is Tyr or His or Gln or Asn orLys or Asp or Glu <400> SEQUENCE: 5 ggcacgagcc gccctctgct gccgacgtgggctgcaggcc gcaggcggtt gccgggcgag 60 caaacggagc gggcggcggg c atg ggc ggcctg cgg ctg ctg gcg gta gcc 111 Met Gly Gly Leu Arg Leu Leu Ala Val Ala1 5 10 ctc acg tgc agc tgc tgg tgg ccg cag ggc ggc cag ggc aag acc ctg159 Leu Thr Cys Ser Cys Trp Trp Pro Gln Gly Gly Gln Gly Lys Thr Leu 1520 25 cgt ggc agc ttc agc agc gcc gcg gcc cgc gac gcc cag ggc cag agc207 Arg Gly Ser Phe Ser Ser Ala Ala Ala Arg Asp Ala Gln Gly Gln Ser 3035 40 atc ggc cat ttc gag ttc cac cga atc aac aac gta gca gtg gct gtt255 Ile Gly His Phe Glu Phe His Arg Ile Asn Asn Val Ala Val Ala Val 4550 55 gga aaa gaa gct aaa ctc tac ctg ttc caa gcc cag gaa tgg ctg aag303 Gly Lys Glu Ala Lys Leu Tyr Leu Phe Gln Ala Gln Glu Trp Leu Lys 6065 70 ctg ctg gag agc agc ccc ggc tac agc tgc agt gag cgg cta gcc cga351 Leu Leu Glu Ser Ser Pro Gly Tyr Ser Cys Ser Glu Arg Leu Ala Arg 7580 85 90 gct cag ctg aca gtg aca gtg acc cag acg gag cac aac ctc aca gtg399 Ala Gln Leu Thr Val Thr Val Thr Gln Thr Glu His Asn Leu Thr Val 95100 105 tcc cag ctg ccc gct ccc cag aca tgg cga gtg ttc tat gcc gac aag447 Ser Gln Leu Pro Ala Pro Gln Thr Trp Arg Val Phe Tyr Ala Asp Lys 110115 120 ttc acc tgc agg gat gac tca nas agc ccc cag ggg gag gag atc ccc495 Phe Thr Cys Arg Asp Asp Ser Xaa Ser Pro Gln Gly Glu Glu Ile Pro 125130 135 ttt gaa atg gtg ctc ctc aac ccg gac gcc gag gga aac ccg ctg gat543 Phe Glu Met Val Leu Leu Asn Pro Asp Ala Glu Gly Asn Pro Leu Asp 140145 150 cat ttt agc gcc aga gag tcc ggg ctc cac gag ttc ttt ttc ctc ctc591 His Phe Ser Ala Arg Glu Ser Gly Leu His Glu Phe Phe Phe Leu Leu 155160 165 170 gtc cta gtg tac ttt gtg act gcg tgc atc tat gcg cag tct ctgtgg 639 Val Leu Val Tyr Phe Val Thr Ala Cys Ile Tyr Ala Gln Ser Leu Trp175 180 185 cag gct atg aag aag gga gga ccc atg cac acc atc tta aag gtcctc 687 Gln Ala Met Lys Lys Gly Gly Pro Met His Thr Ile Leu Lys Val Leu190 195 200 acc act gca ctg ctg ctt caa gct gct tca gcc tta gct aat tacatc 735 Thr Thr Ala Leu Leu Leu Gln Ala Ala Ser Ala Leu Ala Asn Tyr Ile205 210 215 cac ttg tcc agg tac tcc aga gat ggg cta gga gtg cct ctc atagga 783 His Leu Ser Arg Tyr Ser Arg Asp Gly Leu Gly Val Pro Leu Ile Gly220 225 230 agc ctg gca gaa gtt ttt gac att gcc tcc caa att cag atg ctgtac 831 Ser Leu Ala Glu Val Phe Asp Ile Ala Ser Gln Ile Gln Met Leu Tyr235 240 245 250 ctg ctt ctg agc ctg tgt atg ggc tgg aca ata gtg cgg atgaag aag 879 Leu Leu Leu Ser Leu Cys Met Gly Trp Thr Ile Val Arg Met LysLys 255 260 265 tcg cag agc aga ccg ctc cag tgg gac tcg aca ccc gcg tccacg ggc 927 Ser Gln Ser Arg Pro Leu Gln Trp Asp Ser Thr Pro Ala Ser ThrGly 270 275 280 atc gca gtt ttc aty gtc atc aca cag agc att ttg cta ctytgg gag 975 Ile Ala Val Phe Ile Val Ile Thr Gln Ser Ile Leu Leu Leu TrpGlu 285 290 295 cag ttt gaa gac acc agt cac cac agc gca cat tca cac cgcagc tta 1023 Gln Phe Glu Asp Thr Ser His His Ser Ala His Ser His Arg SerLeu 300 305 310 gcc ggg ctc ttg ctg att gtc tta cgg atc tgc ctg gcg ctgtcg ctg 1071 Ala Gly Leu Leu Leu Ile Val Leu Arg Ile Cys Leu Ala Leu SerLeu 315 320 325 330 ggc tgc gga ctt tac cag gtc atc aca gtg gag agg agcgcg ctc aag 1119 Gly Cys Gly Leu Tyr Gln Val Ile Thr Val Glu Arg Ser AlaLeu Lys 335 340 345 aga gag ttc tac atc acg ttt gcc aag ggc tgc atc ctgtgg ttc ttg 1167 Arg Glu Phe Tyr Ile Thr Phe Ala Lys Gly Cys Ile Leu TrpPhe Leu 350 355 360 tgc cag cca gcg ctc gca tgc att gct gtc gct ttt aatgac tac caa 1215 Cys Gln Pro Ala Leu Ala Cys Ile Ala Val Ala Phe Asn AspTyr Gln 365 370 375 aga gat aag ctt atc aca gta ggt gtc atc ctg tgt caggcc gtg gcc 1263 Arg Asp Lys Leu Ile Thr Val Gly Val Ile Leu Cys Gln AlaVal Ala 380 385 390 atg gtc att ctg tac aga ctt ttc ctg tcc cac agt ctttac tgg gag 1311 Met Val Ile Leu Tyr Arg Leu Phe Leu Ser His Ser Leu TyrTrp Glu 395 400 405 410 gtc tcc tcg ctc tcc tca gta acg cta cca ctg accatc tcg tct gca 1359 Val Ser Ser Leu Ser Ser Val Thr Leu Pro Leu Thr IleSer Ser Ala 415 420 425 cac aga ggg cgc cct cat ttc tga tgcttgagttttgtggagag aaccagtgaa 1413 His Arg Gly Arg Pro His Phe 430 tggagaagtgcaataggatc caacgcagca ccgtcttgct gtgcctttgc gtgacagctg 1473 agcggtggaagcagggcgtc ttatttatag aactgaacgt cagcgggctc agcagaaagg 1533 aatagaagctccggagtgaa ctcaaacagt gaacttccca gaaagaatgt tgtttcaagg 1593 tgactgaaacagtttccacg tggaaataaa tgtgaaaagg actgcttaga gtacacgtgg 1653 gccaggtggtcacacctgcg atgcctcgtc actagcaaac tcaggcctga tagtcctaca 1713 gtattcacctagacaatact tgcctgtgcg tgcccagctc gcccagttat gaaatcagcg 1773 ggatgtgctgattttaaaac tacttctttt tatcctttaa agaacgtgca tttcaaatta 1833 taatttaaaggacttgaaag tgaaattact taggaaataa atagaaaata tgttaacagt 1893 taaacgaaaaaaaaaaaaaa aaaaa 1918 <210> SEQ ID NO 6 <211> LENGTH: 433 <212> TYPE:PRT <213> ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: 130 <223> OTHER INFORMATION: Xaa is Tyr orHis or Gln or Asn or Lys or Asp or Glu <400> SEQUENCE: 6 Met Gly Gly LeuArg Leu Leu Ala Val Ala Leu Thr Cys Ser Cys Trp 1 5 10 15 Trp Pro GlnGly Gly Gln Gly Lys Thr Leu Arg Gly Ser Phe Ser Ser 20 25 30 Ala Ala AlaArg Asp Ala Gln Gly Gln Ser Ile Gly His Phe Glu Phe 35 40 45 His Arg IleAsn Asn Val Ala Val Ala Val Gly Lys Glu Ala Lys Leu 50 55 60 Tyr Leu PheGln Ala Gln Glu Trp Leu Lys Leu Leu Glu Ser Ser Pro 65 70 75 80 Gly TyrSer Cys Ser Glu Arg Leu Ala Arg Ala Gln Leu Thr Val Thr 85 90 95 Val ThrGln Thr Glu His Asn Leu Thr Val Ser Gln Leu Pro Ala Pro 100 105 110 GlnThr Trp Arg Val Phe Tyr Ala Asp Lys Phe Thr Cys Arg Asp Asp 115 120 125Ser Xaa Ser Pro Gln Gly Glu Glu Ile Pro Phe Glu Met Val Leu Leu 130 135140 Asn Pro Asp Ala Glu Gly Asn Pro Leu Asp His Phe Ser Ala Arg Glu 145150 155 160 Ser Gly Leu His Glu Phe Phe Phe Leu Leu Val Leu Val Tyr PheVal 165 170 175 Thr Ala Cys Ile Tyr Ala Gln Ser Leu Trp Gln Ala Met LysLys Gly 180 185 190 Gly Pro Met His Thr Ile Leu Lys Val Leu Thr Thr AlaLeu Leu Leu 195 200 205 Gln Ala Ala Ser Ala Leu Ala Asn Tyr Ile His LeuSer Arg Tyr Ser 210 215 220 Arg Asp Gly Leu Gly Val Pro Leu Ile Gly SerLeu Ala Glu Val Phe 225 230 235 240 Asp Ile Ala Ser Gln Ile Gln Met LeuTyr Leu Leu Leu Ser Leu Cys 245 250 255 Met Gly Trp Thr Ile Val Arg MetLys Lys Ser Gln Ser Arg Pro Leu 260 265 270 Gln Trp Asp Ser Thr Pro AlaSer Thr Gly Ile Ala Val Phe Ile Val 275 280 285 Ile Thr Gln Ser Ile LeuLeu Leu Trp Glu Gln Phe Glu Asp Thr Ser 290 295 300 His His Ser Ala HisSer His Arg Ser Leu Ala Gly Leu Leu Leu Ile 305 310 315 320 Val Leu ArgIle Cys Leu Ala Leu Ser Leu Gly Cys Gly Leu Tyr Gln 325 330 335 Val IleThr Val Glu Arg Ser Ala Leu Lys Arg Glu Phe Tyr Ile Thr 340 345 350 PheAla Lys Gly Cys Ile Leu Trp Phe Leu Cys Gln Pro Ala Leu Ala 355 360 365Cys Ile Ala Val Ala Phe Asn Asp Tyr Gln Arg Asp Lys Leu Ile Thr 370 375380 Val Gly Val Ile Leu Cys Gln Ala Val Ala Met Val Ile Leu Tyr Arg 385390 395 400 Leu Phe Leu Ser His Ser Leu Tyr Trp Glu Val Ser Ser Leu SerSer 405 410 415 Val Thr Leu Pro Leu Thr Ile Ser Ser Ala His Arg Gly ArgPro His 420 425 430 Phe <210> SEQ ID NO 7 <211> LENGTH: 2857 <212> TYPE:DNA <213> ORGANISM: Oryctolagus cuniculus <220> FEATURE: <221> NAME/KEY:CDS <222> LOCATION: (415)..(2199) <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (745)..(747) <223> OTHER INFORMATION: Xaais Met or Leu <400> SEQUENCE: 7 gtgttactgt gtttcactaa atgtttgaaggctgtcggac tttttgaatc atatgatctc 60 ctgaaagtag ttcacattgt tcagttcgtttttatattaa aacttgggac tgcatttttt 120 atggttttgt ttcaaaagcc attttcttctgggaaaacta ttaccaaaca ccagtggatc 180 acaatattta aacatgcagt tgccgggtgtatcatttcac tcttgtggtt ttttggcctt 240 accctttgtg gaccactaag gactttgctgctgtttgaac acagtgaaat tgttgtcatc 300 tcgctcctca gtgttttgtt caccagttctggaggaggac cagcaaagac aagaggggct 360 gcttttttca tcattgctgt gatctgtttattgctttttg acaatgatga tctc atg 417 Met 1 gct aaa atg gca gaa cac cct gaagga cat cat gac agt gct cta act 465 Ala Lys Met Ala Glu His Pro Glu GlyHis His Asp Ser Ala Leu Thr 5 10 15 cac atg ctt tac aca gcc att gcc ttctta ggt gtg gca gat cac aag 513 His Met Leu Tyr Thr Ala Ile Ala Phe LeuGly Val Ala Asp His Lys 20 25 30 ggt gga gta ttg ttg cta gta ctg gct ttgtgt tgt aaa gtt ggt ttt 561 Gly Gly Val Leu Leu Leu Val Leu Ala Leu CysCys Lys Val Gly Phe 35 40 45 cac aca gct tcc aga aaa ctc tct ata gat gttggg gga gcc aaa cgt 609 His Thr Ala Ser Arg Lys Leu Ser Ile Asp Val GlyGly Ala Lys Arg 50 55 60 65 ctt caa gct tta tcc cat ctt gtt tct gtg cttctc ttg tgc cca tgg 657 Leu Gln Ala Leu Ser His Leu Val Ser Val Leu LeuLeu Cys Pro Trp 70 75 80 gtc att gtt ctt tct atg aca act gag agt aaa gttgag tct tgg ttt 705 Val Ile Val Leu Ser Met Thr Thr Glu Ser Lys Val GluSer Trp Phe 85 90 95 tct ctc att atg cct ttc acg atg gtt att ttt ttt gtcwtg atc ctg 753 Ser Leu Ile Met Pro Phe Thr Met Val Ile Phe Phe Val XaaIle Leu 100 105 110 gat ttc tac gtg gat tcc att tgt tca gtc aaa atg gaagtt tcc aaa 801 Asp Phe Tyr Val Asp Ser Ile Cys Ser Val Lys Met Glu ValSer Lys 115 120 125 tgt gcc cgc tat gga tcc ttg ccc att ttt att agt gctctc ctt ttt 849 Cys Ala Arg Tyr Gly Ser Leu Pro Ile Phe Ile Ser Ala LeuLeu Phe 130 135 140 145 gga aat ttc tgg acc cac ccc ata act gac cag cttcgg gca atg agc 897 Gly Asn Phe Trp Thr His Pro Ile Thr Asp Gln Leu ArgAla Met Ser 150 155 160 aga gca gca cac cag ggg agc acg gaa cac gtt ctgtct gga gga gtg 945 Arg Ala Ala His Gln Gly Ser Thr Glu His Val Leu SerGly Gly Val 165 170 175 gtc gtg agc gca gtg ttc ttc atc ttg tct gcc aacatc ctg tca tct 993 Val Val Ser Ala Val Phe Phe Ile Leu Ser Ala Asn IleLeu Ser Ser 180 185 190 cct tcg aag agg ggg cag aag ggc acc ctg att ggatac tct cct gaa 1041 Pro Ser Lys Arg Gly Gln Lys Gly Thr Leu Ile Gly TyrSer Pro Glu 195 200 205 gga gca cct ctt tac aac ttc atg ggg gat gct tttcag cac agc tca 1089 Gly Ala Pro Leu Tyr Asn Phe Met Gly Asp Ala Phe GlnHis Ser Ser 210 215 220 225 cag tcc gtg cct cgg ttt att aag gaa tcg ctgaaa cag att ctt gag 1137 Gln Ser Val Pro Arg Phe Ile Lys Glu Ser Leu LysGln Ile Leu Glu 230 235 240 gag agt gac tct agg cag atc ttt tac ttc ttgtgc ttg aat ctg ctt 1185 Glu Ser Asp Ser Arg Gln Ile Phe Tyr Phe Leu CysLeu Asn Leu Leu 245 250 255 ttt acc ttt gtg gaa tta ttc tat gga gtg ctgacg aat agt ctg ggt 1233 Phe Thr Phe Val Glu Leu Phe Tyr Gly Val Leu ThrAsn Ser Leu Gly 260 265 270 ctg atc tca gat ggc ttt cac atg ctc ttt gactgc tct gcc ttg gtc 1281 Leu Ile Ser Asp Gly Phe His Met Leu Phe Asp CysSer Ala Leu Val 275 280 285 atg gga ctt ttt gct gcc ctg atg agt aga tggaaa gca act cgg att 1329 Met Gly Leu Phe Ala Ala Leu Met Ser Arg Trp LysAla Thr Arg Ile 290 295 300 305 ttc tcc tac ggg tat ggc cga ata gaa attctt tct gga ttt att aat 1377 Phe Ser Tyr Gly Tyr Gly Arg Ile Glu Ile LeuSer Gly Phe Ile Asn 310 315 320 gga ctt ttt cta ata gta ata gct ttt tttgtg ttt atg gag tca gtt 1425 Gly Leu Phe Leu Ile Val Ile Ala Phe Phe ValPhe Met Glu Ser Val 325 330 335 gcc aga ttg att gat cct ccg gaa tta gacaca aac atg cta aca cca 1473 Ala Arg Leu Ile Asp Pro Pro Glu Leu Asp ThrAsn Met Leu Thr Pro 340 345 350 gtg tca gtt gga ggg ctg ata gta aac cttatt ggt atc tgt gcc ttt 1521 Val Ser Val Gly Gly Leu Ile Val Asn Leu IleGly Ile Cys Ala Phe 355 360 365 agc cac gcc cat aat cac acc cat gga tcttcc caa gga agc tgt cac 1569 Ser His Ala His Asn His Thr His Gly Ser SerGln Gly Ser Cys His 370 375 380 385 tca tcc gat cac agc cat tca cac cacatg cat gga cac agt gac cat 1617 Ser Ser Asp His Ser His Ser His His MetHis Gly His Ser Asp His 390 395 400 gga cat ggt cac agc cat gga tcc ccaggc ggc ggc atg aat gct aac 1665 Gly His Gly His Ser His Gly Ser Pro GlyGly Gly Met Asn Ala Asn 405 410 415 atg agg ggt gtg ttt ttc cat gtt ttggca gac acg ctt ggc agt att 1713 Met Arg Gly Val Phe Phe His Val Leu AlaAsp Thr Leu Gly Ser Ile 420 425 430 ggt gtg att gta ttt aca gtt ttt atagag cag ttt ggg tgg ttc att 1761 Gly Val Ile Val Phe Thr Val Phe Ile GluGln Phe Gly Trp Phe Ile 435 440 445 gcg gat ccc ctc tgt tct ctc ttt attgct gta tta ata ttt ctc agt 1809 Ala Asp Pro Leu Cys Ser Leu Phe Ile AlaVal Leu Ile Phe Leu Ser 450 455 460 465 gtt gtc cca ctg atc aaa gat gcctgt cag gtt cta ctt ttg aga ctg 1857 Val Val Pro Leu Ile Lys Asp Ala CysGln Val Leu Leu Leu Arg Leu 470 475 480 cca cca gag tat gaa aaa gaa ctacat att gct tta gaa aag ata caa 1905 Pro Pro Glu Tyr Glu Lys Glu Leu HisIle Ala Leu Glu Lys Ile Gln 485 490 495 aaa att gar gga tta ata tca taccga gat cct cat ttc tgg cgc cat 1953 Lys Ile Glu Gly Leu Ile Ser Tyr ArgAsp Pro His Phe Trp Arg His 500 505 510 tct gcc agt att gtg gca gga acaatt cat ata caa gtg aca tct gat 2001 Ser Ala Ser Ile Val Ala Gly Thr IleHis Ile Gln Val Thr Ser Asp 515 520 525 gtg cta gaa caa aga ata gta cagcag gtt aca gga ata ctt aaa gat 2049 Val Leu Glu Gln Arg Ile Val Gln GlnVal Thr Gly Ile Leu Lys Asp 530 535 540 545 gca gga gta aac aat tta acaatt caa gta gaa aaa gaa gca tac ttt 2097 Ala Gly Val Asn Asn Leu Thr IleGln Val Glu Lys Glu Ala Tyr Phe 550 555 560 caa cat atg tct ggc cta agtact gga ttt cat gat gtt ctg gct atg 2145 Gln His Met Ser Gly Leu Ser ThrGly Phe His Asp Val Leu Ala Met 565 570 575 aca aaa caa atg gag tcc atgaaa tac tgc aag gat ggc act tac att 2193 Thr Lys Gln Met Glu Ser Met LysTyr Cys Lys Asp Gly Thr Tyr Ile 580 585 590 atg tga gagaactcacagattacccc cgatgtgagc agtgaagatt cagtgactca 2249 Met 595 gtgttgtaacattgccagca ggacagaaac tgcgtgtaat tgtacagaga ttttaaagct 2309 ccctattcttggatcaagga ctctttccta aaggaaattt aaatattgat tgaaacattg 2369 atcacacagtaaaatagtga tttgagttat gtattttaaa tgactcttac aatttgaaca 2429 taatgtgtctcatcatcttc agaaatggac acaatgatgg attctaatga agaccaaaag 2489 tacttctgtgtttcctttct gtcagaaagc atctccattg taaatatgta tttacatgtt 2549 tattacaaagatccaaatga aaaattttta gtccattttt tgcatagcct aaagataaaa 2609 taggaataaaagttctatat ttatgaattt tctgtacata aaactggttt ctaattataa 2669 ctgaagtccactgggtaaaa tctgtattgc caccttaaat gtaaactaaa ttatttgaga 2729 gaaacttcaaccactgatat gacataagca gtgagaacag ggagtctata acattacagt 2789 tttggatgttaccaaaacca accactctgt aaaataaatt ttttactttt gtcaaaaaaa 2849 aaaaaaaa2857 <210> SEQ ID NO 8 <211> LENGTH: 594 <212> TYPE: PRT <213> ORGANISM:Oryctolagus cuniculus <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: 111 <223> OTHER INFORMATION: Xaa is Met or Leu <400> SEQUENCE:8 Met Ala Lys Met Ala Glu His Pro Glu Gly His His Asp Ser Ala Leu 1 5 1015 Thr His Met Leu Tyr Thr Ala Ile Ala Phe Leu Gly Val Ala Asp His 20 2530 Lys Gly Gly Val Leu Leu Leu Val Leu Ala Leu Cys Cys Lys Val Gly 35 4045 Phe His Thr Ala Ser Arg Lys Leu Ser Ile Asp Val Gly Gly Ala Lys 50 5560 Arg Leu Gln Ala Leu Ser His Leu Val Ser Val Leu Leu Leu Cys Pro 65 7075 80 Trp Val Ile Val Leu Ser Met Thr Thr Glu Ser Lys Val Glu Ser Trp 8590 95 Phe Ser Leu Ile Met Pro Phe Thr Met Val Ile Phe Phe Val Xaa Ile100 105 110 Leu Asp Phe Tyr Val Asp Ser Ile Cys Ser Val Lys Met Glu ValSer 115 120 125 Lys Cys Ala Arg Tyr Gly Ser Leu Pro Ile Phe Ile Ser AlaLeu Leu 130 135 140 Phe Gly Asn Phe Trp Thr His Pro Ile Thr Asp Gln LeuArg Ala Met 145 150 155 160 Ser Arg Ala Ala His Gln Gly Ser Thr Glu HisVal Leu Ser Gly Gly 165 170 175 Val Val Val Ser Ala Val Phe Phe Ile LeuSer Ala Asn Ile Leu Ser 180 185 190 Ser Pro Ser Lys Arg Gly Gln Lys GlyThr Leu Ile Gly Tyr Ser Pro 195 200 205 Glu Gly Ala Pro Leu Tyr Asn PheMet Gly Asp Ala Phe Gln His Ser 210 215 220 Ser Gln Ser Val Pro Arg PheIle Lys Glu Ser Leu Lys Gln Ile Leu 225 230 235 240 Glu Glu Ser Asp SerArg Gln Ile Phe Tyr Phe Leu Cys Leu Asn Leu 245 250 255 Leu Phe Thr PheVal Glu Leu Phe Tyr Gly Val Leu Thr Asn Ser Leu 260 265 270 Gly Leu IleSer Asp Gly Phe His Met Leu Phe Asp Cys Ser Ala Leu 275 280 285 Val MetGly Leu Phe Ala Ala Leu Met Ser Arg Trp Lys Ala Thr Arg 290 295 300 IlePhe Ser Tyr Gly Tyr Gly Arg Ile Glu Ile Leu Ser Gly Phe Ile 305 310 315320 Asn Gly Leu Phe Leu Ile Val Ile Ala Phe Phe Val Phe Met Glu Ser 325330 335 Val Ala Arg Leu Ile Asp Pro Pro Glu Leu Asp Thr Asn Met Leu Thr340 345 350 Pro Val Ser Val Gly Gly Leu Ile Val Asn Leu Ile Gly Ile CysAla 355 360 365 Phe Ser His Ala His Asn His Thr His Gly Ser Ser Gln GlySer Cys 370 375 380 His Ser Ser Asp His Ser His Ser His His Met His GlyHis Ser Asp 385 390 395 400 His Gly His Gly His Ser His Gly Ser Pro GlyGly Gly Met Asn Ala 405 410 415 Asn Met Arg Gly Val Phe Phe His Val LeuAla Asp Thr Leu Gly Ser 420 425 430 Ile Gly Val Ile Val Phe Thr Val PheIle Glu Gln Phe Gly Trp Phe 435 440 445 Ile Ala Asp Pro Leu Cys Ser LeuPhe Ile Ala Val Leu Ile Phe Leu 450 455 460 Ser Val Val Pro Leu Ile LysAsp Ala Cys Gln Val Leu Leu Leu Arg 465 470 475 480 Leu Pro Pro Glu TyrGlu Lys Glu Leu His Ile Ala Leu Glu Lys Ile 485 490 495 Gln Lys Ile GluGly Leu Ile Ser Tyr Arg Asp Pro His Phe Trp Arg 500 505 510 His Ser AlaSer Ile Val Ala Gly Thr Ile His Ile Gln Val Thr Ser 515 520 525 Asp ValLeu Glu Gln Arg Ile Val Gln Gln Val Thr Gly Ile Leu Lys 530 535 540 AspAla Gly Val Asn Asn Leu Thr Ile Gln Val Glu Lys Glu Ala Tyr 545 550 555560 Phe Gln His Met Ser Gly Leu Ser Thr Gly Phe His Asp Val Leu Ala 565570 575 Met Thr Lys Gln Met Glu Ser Met Lys Tyr Cys Lys Asp Gly Thr Tyr580 585 590 Ile Met <210> SEQ ID NO 9 <211> LENGTH: 2519 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(1785) <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (289)..(291) <223> OTHER INFORMATION: Xaa is Phe or Leu <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1015)..(1017)<223> OTHER INFORMATION: Xaa is Lys or Arg <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1492)..(1494) <223> OTHERINFORMATION: n is a or g or c or t Xaa is Gln or Lys or Glu <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1540)..(1542),(1582)..(1584), (1693)..(1695) <223> OTHER INFORMATION: Xaa is Phe orSer <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(1609)..(1611) <223> OTHER INFORMATION: Xaa is Gln or Arg <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: 2493, 2516 <223> OTHERINFORMATION: n is a or g or c or t <400> SEQUENCE: 9 atg gct aaa atg gctgaa cac cct gaa gga cat cat gac agt gct cta 48 Met Ala Lys Met Ala GluHis Pro Glu Gly His His Asp Ser Ala Leu 1 5 10 15 act cat atg ctt tacaca gcc att gcc ttc tta ggt gtg gca gat cac 96 Thr His Met Leu Tyr ThrAla Ile Ala Phe Leu Gly Val Ala Asp His 20 25 30 aag ggt gga gta tta ttgcta gta ctg gct ttg tgt tgt aaa gtt ggt 144 Lys Gly Gly Val Leu Leu LeuVal Leu Ala Leu Cys Cys Lys Val Gly 35 40 45 ttt cat aca gct tcc aga aagctc tct gtc gac gtt ggt gga gct aaa 192 Phe His Thr Ala Ser Arg Lys LeuSer Val Asp Val Gly Gly Ala Lys 50 55 60 cgt ctt caa gct tta tct cat cttgtt tct gtg ctt ctc ttg tgc cca 240 Arg Leu Gln Ala Leu Ser His Leu ValSer Val Leu Leu Leu Cys Pro 65 70 75 80 tgg gtc att gtt ctt tct gtg acaact gag agt aaa gtg gag tct tgg 288 Trp Val Ile Val Leu Ser Val Thr ThrGlu Ser Lys Val Glu Ser Trp 85 90 95 ytt tct ctc att atg cct ttt gca acggtt atc ttt ttt gtc atg atc 336 Xaa Ser Leu Ile Met Pro Phe Ala Thr ValIle Phe Phe Val Met Ile 100 105 110 ctg gat ttc tac gtg gat tcc att tgttca gtc aaa atg gaa gtt tcc 384 Leu Asp Phe Tyr Val Asp Ser Ile Cys SerVal Lys Met Glu Val Ser 115 120 125 aaa tgt gct cgt tat gga tcc ttt cccatt ttt att agt gct ctc ctt 432 Lys Cys Ala Arg Tyr Gly Ser Phe Pro IlePhe Ile Ser Ala Leu Leu 130 135 140 ttt gga aat ttt tgg aca cat cca ataaca gac cag ctt cgg gct atg 480 Phe Gly Asn Phe Trp Thr His Pro Ile ThrAsp Gln Leu Arg Ala Met 145 150 155 160 aac aaa gca gca cac cag gag agcact gaa cac gtc ctg tct gga gga 528 Asn Lys Ala Ala His Gln Glu Ser ThrGlu His Val Leu Ser Gly Gly 165 170 175 gtg gta gtg agt gct ata ttc ttcatt ttg tct gcc aat atc tta tca 576 Val Val Val Ser Ala Ile Phe Phe IleLeu Ser Ala Asn Ile Leu Ser 180 185 190 tct ccc tct aag aga gga caa aaaggt acc ctt att gga tat tct cct 624 Ser Pro Ser Lys Arg Gly Gln Lys GlyThr Leu Ile Gly Tyr Ser Pro 195 200 205 gaa gga aca cct ctt tat aac ttcatg ggt gat gct ttt cag cat agc 672 Glu Gly Thr Pro Leu Tyr Asn Phe MetGly Asp Ala Phe Gln His Ser 210 215 220 tct caa tcg atc cct agg ttt attaag gaa tca cta aaa caa att ctt 720 Ser Gln Ser Ile Pro Arg Phe Ile LysGlu Ser Leu Lys Gln Ile Leu 225 230 235 240 gag gag agt gac tct agg cagatc ttt tac ttc ttg tgc ttg aat ctg 768 Glu Glu Ser Asp Ser Arg Gln IlePhe Tyr Phe Leu Cys Leu Asn Leu 245 250 255 ctt ttt acc ttt gtg gaa ttattc tat ggc gtg ctg acc aat agt ctg 816 Leu Phe Thr Phe Val Glu Leu PheTyr Gly Val Leu Thr Asn Ser Leu 260 265 270 ggc ctg atc tcg gat gga ttccac atg ctt ttt gac tgc tct gct tta 864 Gly Leu Ile Ser Asp Gly Phe HisMet Leu Phe Asp Cys Ser Ala Leu 275 280 285 gtc atg gga ctt ttt gct gccctg atg agt agg tgg aaa gcc act cgg 912 Val Met Gly Leu Phe Ala Ala LeuMet Ser Arg Trp Lys Ala Thr Arg 290 295 300 att ttc tcc tat ggg tac ggccga ata gaa att ctg tct gga ttt att 960 Ile Phe Ser Tyr Gly Tyr Gly ArgIle Glu Ile Leu Ser Gly Phe Ile 305 310 315 320 aat gga ctt ttt cta atagta ata gcg ttt ttt gtg ttt atg gag tca 1008 Asn Gly Leu Phe Leu Ile ValIle Ala Phe Phe Val Phe Met Glu Ser 325 330 335 gtg gct ara ttg att gatcct cca gaa tta gac act cac atg tta aca 1056 Val Ala Xaa Leu Ile Asp ProPro Glu Leu Asp Thr His Met Leu Thr 340 345 350 cca gty tca gtt gga gggctg ata gta aac ctt att ggt atc tgt gcc 1104 Pro Val Ser Val Gly Gly LeuIle Val Asn Leu Ile Gly Ile Cys Ala 355 360 365 ttt agc cat gcc cat agccat gcc cat gga gct tct caa gga agc tgt 1152 Phe Ser His Ala His Ser HisAla His Gly Ala Ser Gln Gly Ser Cys 370 375 380 cac tca tct gat cac agccat tca cay cat atg cat gga cac agt gac 1200 His Ser Ser Asp His Ser HisSer His His Met His Gly His Ser Asp 385 390 395 400 cat ggg cat ggt cacagc cac gga tct gcg ggt gga ggc atg aat gct 1248 His Gly His Gly His SerHis Gly Ser Ala Gly Gly Gly Met Asn Ala 405 410 415 aac atg agg ggt gtattt yta cat gtt ttg gca gat acw ctt ggc agc 1296 Asn Met Arg Gly Val PheLeu His Val Leu Ala Asp Thr Leu Gly Ser 420 425 430 att ggt gtg att gtatcc aca gtt ttt ata gag cag ttt gga tgg ttc 1344 Ile Gly Val Ile Val SerThr Val Phe Ile Glu Gln Phe Gly Trp Phe 435 440 445 atc gct gac cca ctctgt tct ctt ttt att gct ata tta ata ttt ctc 1392 Ile Ala Asp Pro Leu CysSer Leu Phe Ile Ala Ile Leu Ile Phe Leu 450 455 460 agt gtt gtt cca ctgatt aaa gat gcc tgc cag gtt tta ctc ctg aga 1440 Ser Val Val Pro Leu IleLys Asp Ala Cys Gln Val Leu Leu Leu Arg 465 470 475 480 ttg cca cca gaatat gga aaa gaa cta cat att gct tta gaa aag ata 1488 Leu Pro Pro Glu TyrGly Lys Glu Leu His Ile Ala Leu Glu Lys Ile 485 490 495 cag naa att gaagga tta ata tca tac cga gac cct cat ttt tgg cgt 1536 Gln Xaa Ile Glu GlyLeu Ile Ser Tyr Arg Asp Pro His Phe Trp Arg 500 505 510 cat tyt gct agtatt gtg gca gga aca att cat ata cag gtg aca tyt 1584 His Xaa Ala Ser IleVal Ala Gly Thr Ile His Ile Gln Val Thr Xaa 515 520 525 gat gtg cta gaacaa aga ata gta crg cag gtt aca gga ata ctt aaa 1632 Asp Val Leu Glu GlnArg Ile Val Xaa Gln Val Thr Gly Ile Leu Lys 530 535 540 gat gct gga gtaaac aat tta aca att caa gtg gaa aag gag gca tac 1680 Asp Ala Gly Val AsnAsn Leu Thr Ile Gln Val Glu Lys Glu Ala Tyr 545 550 555 560 ttt caa catatg tyt ggc cta agt act gga ttt cat gat gtt ctg gct 1728 Phe Gln His MetXaa Gly Leu Ser Thr Gly Phe His Asp Val Leu Ala 565 570 575 atg aca aaacaa atg gaa tcc atg aaa tac tgc aaa gat ggt act tac 1776 Met Thr Lys GlnMet Glu Ser Met Lys Tyr Cys Lys Asp Gly Thr Tyr 580 585 590 atc atg tgagataactcaa gaattacccc tggagaataa acaatgaaga 1825 Ile Met 595 ttaaatgactcagtatttgt aatattgcca gaaggataaa aattacacat taactgtaca 1885 gaaacagagttccctactac tggatcaagg aatctttctt gaaggaaatt taaatacaga 1945 atgaaacattaatggtaaaa gtggagtaat tatttaaatt atgtgtataa aaggaatcaa 2005 attttgagtaaacatgatgt attacatcat cttcaaaaat agatatgatg gattctagtg 2065 aagaccaaaattacttctgt ttactttcta tcaggaagca tctccattgt aaatatgtat 2125 ttacatgtttattacaaaga cccaaatgaa aaatttttag tccatttttt gcatagccta 2185 aagataaaataggaataaaa gttctatatt tatgggattt tctgtatata aaactggttt 2245 ctaattataacttaagtcca ttaagtaaaa tctgtattgc cactttaaat gtaaactaaa 2305 ttatttgggagaaacttcaa ccactgatat gagataagca atgagaatag ggaagtgtat 2365 aacatcacagtttttgatgt attacaaaaa tcaaccactt tataaaataa atttttttta 2425 cttttggtaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaag cggccgctga 2485 attctagntagaattcagcg gccgctgaat ncta 2519 <210> SEQ ID NO 10 <211> LENGTH: 594<212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: 97 <223> OTHER INFORMATION: Xaais Phe or Leu <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: 339 <223> OTHER INFORMATION: Xaa is Lys or Arg <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: 498 <223> OTHERINFORMATION: Xaa is Gln or Lys or Glu <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: 514, 528, 565 <223> OTHER INFORMATION: Xaais Phe or Ser <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: 537 <223> OTHER INFORMATION: Xaa is Gln or Arg <400> SEQUENCE:10 Met Ala Lys Met Ala Glu His Pro Glu Gly His His Asp Ser Ala Leu 1 510 15 Thr His Met Leu Tyr Thr Ala Ile Ala Phe Leu Gly Val Ala Asp His 2025 30 Lys Gly Gly Val Leu Leu Leu Val Leu Ala Leu Cys Cys Lys Val Gly 3540 45 Phe His Thr Ala Ser Arg Lys Leu Ser Val Asp Val Gly Gly Ala Lys 5055 60 Arg Leu Gln Ala Leu Ser His Leu Val Ser Val Leu Leu Leu Cys Pro 6570 75 80 Trp Val Ile Val Leu Ser Val Thr Thr Glu Ser Lys Val Glu Ser Trp85 90 95 Xaa Ser Leu Ile Met Pro Phe Ala Thr Val Ile Phe Phe Val Met Ile100 105 110 Leu Asp Phe Tyr Val Asp Ser Ile Cys Ser Val Lys Met Glu ValSer 115 120 125 Lys Cys Ala Arg Tyr Gly Ser Phe Pro Ile Phe Ile Ser AlaLeu Leu 130 135 140 Phe Gly Asn Phe Trp Thr His Pro Ile Thr Asp Gln LeuArg Ala Met 145 150 155 160 Asn Lys Ala Ala His Gln Glu Ser Thr Glu HisVal Leu Ser Gly Gly 165 170 175 Val Val Val Ser Ala Ile Phe Phe Ile LeuSer Ala Asn Ile Leu Ser 180 185 190 Ser Pro Ser Lys Arg Gly Gln Lys GlyThr Leu Ile Gly Tyr Ser Pro 195 200 205 Glu Gly Thr Pro Leu Tyr Asn PheMet Gly Asp Ala Phe Gln His Ser 210 215 220 Ser Gln Ser Ile Pro Arg PheIle Lys Glu Ser Leu Lys Gln Ile Leu 225 230 235 240 Glu Glu Ser Asp SerArg Gln Ile Phe Tyr Phe Leu Cys Leu Asn Leu 245 250 255 Leu Phe Thr PheVal Glu Leu Phe Tyr Gly Val Leu Thr Asn Ser Leu 260 265 270 Gly Leu IleSer Asp Gly Phe His Met Leu Phe Asp Cys Ser Ala Leu 275 280 285 Val MetGly Leu Phe Ala Ala Leu Met Ser Arg Trp Lys Ala Thr Arg 290 295 300 IlePhe Ser Tyr Gly Tyr Gly Arg Ile Glu Ile Leu Ser Gly Phe Ile 305 310 315320 Asn Gly Leu Phe Leu Ile Val Ile Ala Phe Phe Val Phe Met Glu Ser 325330 335 Val Ala Xaa Leu Ile Asp Pro Pro Glu Leu Asp Thr His Met Leu Thr340 345 350 Pro Val Ser Val Gly Gly Leu Ile Val Asn Leu Ile Gly Ile CysAla 355 360 365 Phe Ser His Ala His Ser His Ala His Gly Ala Ser Gln GlySer Cys 370 375 380 His Ser Ser Asp His Ser His Ser His His Met His GlyHis Ser Asp 385 390 395 400 His Gly His Gly His Ser His Gly Ser Ala GlyGly Gly Met Asn Ala 405 410 415 Asn Met Arg Gly Val Phe Leu His Val LeuAla Asp Thr Leu Gly Ser 420 425 430 Ile Gly Val Ile Val Ser Thr Val PheIle Glu Gln Phe Gly Trp Phe 435 440 445 Ile Ala Asp Pro Leu Cys Ser LeuPhe Ile Ala Ile Leu Ile Phe Leu 450 455 460 Ser Val Val Pro Leu Ile LysAsp Ala Cys Gln Val Leu Leu Leu Arg 465 470 475 480 Leu Pro Pro Glu TyrGly Lys Glu Leu His Ile Ala Leu Glu Lys Ile 485 490 495 Gln Xaa Ile GluGly Leu Ile Ser Tyr Arg Asp Pro His Phe Trp Arg 500 505 510 His Xaa AlaSer Ile Val Ala Gly Thr Ile His Ile Gln Val Thr Xaa 515 520 525 Asp ValLeu Glu Gln Arg Ile Val Xaa Gln Val Thr Gly Ile Leu Lys 530 535 540 AspAla Gly Val Asn Asn Leu Thr Ile Gln Val Glu Lys Glu Ala Tyr 545 550 555560 Phe Gln His Met Xaa Gly Leu Ser Thr Gly Phe His Asp Val Leu Ala 565570 575 Met Thr Lys Gln Met Glu Ser Met Lys Tyr Cys Lys Asp Gly Thr Tyr580 585 590 Ile Met <210> SEQ ID NO 11 <211> LENGTH: 178 <212> TYPE: DNA<213> ORGANISM: Oryctolagus cuniculus <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: 54, 68, 97 <223> OTHER INFORMATION: n is aor g or c or t <400> SEQUENCE: 11 gttttttttt tttttcatac atttggtatgaaacatcgaa tagcaaaagc agancatgtt 60 tctgtatnac tgcatttaag cagtaccaaaactgaanaaa ggtaataact gaaatgtttt 120 aaaatacatg taaacaataa actttcaggaaattctgttg ttaaaaaaaa aaaaaaac 178 <210> SEQ ID NO 12 <211> LENGTH: 167<212> TYPE: DNA <213> ORGANISM: Oryctolagus cuniculus <400> SEQUENCE: 12cttgattgcc accttaaatg taaactaaat tatttgagag aaacttcaac cactgatatg 60acataagcag tgagaacagg gagtctataa cattacagtt ttggatgtta ccaaaaccaa 120ccactctgta aaataaattt tttacttttg taaaaaaaaa aaaaaac 167 <210> SEQ ID NO13 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence:an artificially synthesized primer sequence <220> FEATURE:<221> NAME/KEY: primer_bind <222> LOCATION: (1)..(24) <400> SEQUENCE: 13tcatacattt ggtatgaaac atcg 24 <210> SEQ ID NO 14 <211> LENGTH: 24 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:an artificiallysynthesized primer sequence <220> FEATURE: <221> NAME/KEY: primer_bind<222> LOCATION: (1)..(24) <400> SEQUENCE: 14 tttttttaac aacagaattt cctg24 <210> SEQ ID NO 15 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence:an artificially synthesized primer sequence <220>FEATURE: <221> NAME/KEY: primer_bind <222> LOCATION: (1)..(27) <400>SEQUENCE: 15 aatttcctga aagtttattg tttacat 27 <210> SEQ ID NO 16 <211>LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:anartificially synthesized primer sequence <220> FEATURE: <221> NAME/KEY:primer_bind <222> LOCATION: (1)..(25) <400> SEQUENCE: 16 gaaacatgctctgcttttgc tattc 25 <210> SEQ ID NO 17 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:an artificiallysynthesized primer sequence <220> FEATURE: <221> NAME/KEY: primer_bind<222> LOCATION: (1)..(27) <400> SEQUENCE: 17 atccctgcta agttgtggtgtgaatgg 27 <210> SEQ ID NO 18 <211> LENGTH: 27 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence:an artificially synthesized primersequence <220> FEATURE: <221> NAME/KEY: primer_bind <222> LOCATION:(1)..(27) <400> SEQUENCE: 18 tctgctttga gacttcttca tcctgac 27 <210> SEQID NO 19 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence:an artificially synthesized primer sequence <220>FEATURE: <221> NAME/KEY: primer_bind <222> LOCATION: (1)..(27) <400>SEQUENCE: 19 ccatcctaat acgactcact atagggc 27 <210> SEQ ID NO 20 <211>LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:anartificially synthesized primer sequence <220> FEATURE: <221> NAME/KEY:primer_bind <222> LOCATION: (1)..(23) <400> SEQUENCE: 20 actcactatagggctcgagc ggc 23 <210> SEQ ID NO 21 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:an artificiallysynthesized primer sequence <220> FEATURE: <221> NAME/KEY: primer_bind<222> LOCATION: (1)..(22) <400> SEQUENCE: 21 ttgattgcca ccttaaatgt aa 22<210> SEQ ID NO 22 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence:an artificially synthesized primer sequence <220>FEATURE: <221> NAME/KEY: primer_bind <222> LOCATION: (1)..(22) <400>SEQUENCE: 22 gtggttggtt ttggtaacat cc 22 <210> SEQ ID NO 23 <211>LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:anartificially synthesized primer sequence <220> FEATURE: <221> NAME/KEY:primer_bind <222> LOCATION: (1)..(25) <400> SEQUENCE: 23 gttatagactccctgttctc actgc 25 <210> SEQ ID NO 24 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:an artificiallysynthesized primer sequence <220> FEATURE: <221> NAME/KEY: primer_bind<222> LOCATION: (1)..(25) <400> SEQUENCE: 24 agactccctg ttctcactgc ttatg25 <210> SEQ ID NO 25 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence:an artificially synthesized primer sequence <220>FEATURE: <221> NAME/KEY: primer_bind <222> LOCATION: (1)..(27) <400>SEQUENCE: 25 atcttcactg ctcacatcgg gggtaat 27 <210> SEQ ID NO 26 <211>LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:anartificially synthesized primer sequence <220> FEATURE: <221> NAME/KEY:primer_bind <222> LOCATION: (1)..(23) <400> SEQUENCE: 26 tcactgctcacatcgggggt aat 23 <210> SEQ ID NO 27 <211> LENGTH: 1703 <212> TYPE: DNA<213> ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (10)..(1701) <400> SEQUENCE: 27 gatgatctc atg gca aag atg gctgaa cac ccg gaa gga cat cat gat agt 51 Met Ala Lys Met Ala Glu His ProGlu Gly His His Asp Ser 1 5 10 gct cta act cac atg ctc tat aca gcc attgcc ttt tta ggg gtg gca 99 Ala Leu Thr His Met Leu Tyr Thr Ala Ile AlaPhe Leu Gly Val Ala 15 20 25 30 gat cac aag ggt gga gta ctc ttg ctg gtgctg gct tta tgt tgt aaa 147 Asp His Lys Gly Gly Val Leu Leu Leu Val LeuAla Leu Cys Cys Lys 35 40 45 gtt ggt ttt cat acg gct tcc aga aag ctc tctata gat gtt ggt gga 195 Val Gly Phe His Thr Ala Ser Arg Lys Leu Ser IleAsp Val Gly Gly 50 55 60 gct aag cgc ctt cag gcc tta tct cag ctt gtt tctgtg ttt ctc ctg 243 Ala Lys Arg Leu Gln Ala Leu Ser Gln Leu Val Ser ValPhe Leu Leu 65 70 75 tgc cca tgg gtg att gtc ctt tct gtg aca act gaa agtaag gtt gag 291 Cys Pro Trp Val Ile Val Leu Ser Val Thr Thr Glu Ser LysVal Glu 80 85 90 tct tgg ttc tct ctc atc atg cct ttc acc aca gtc atc tttttt gtc 339 Ser Trp Phe Ser Leu Ile Met Pro Phe Thr Thr Val Ile Phe PheVal 95 100 105 110 atg atc ctg gat ttc tat atg gat tct gtt tgt tca gtcaaa atg gac 387 Met Ile Leu Asp Phe Tyr Met Asp Ser Val Cys Ser Val LysMet Asp 115 120 125 gtg tcc aaa tgt gcc cgc tat ggg tcc ttt ccc att tttatt agt gcc 435 Val Ser Lys Cys Ala Arg Tyr Gly Ser Phe Pro Ile Phe IleSer Ala 130 135 140 ctc ctg ttt cga aat ttc tgg aca cac cca ata aca gaccaa ctc cgg 483 Leu Leu Phe Arg Asn Phe Trp Thr His Pro Ile Thr Asp GlnLeu Arg 145 150 155 gct atg aac aga gca gca cac cag gag agc aca gaa cacgtc ctg tct 531 Ala Met Asn Arg Ala Ala His Gln Glu Ser Thr Glu His ValLeu Ser 160 165 170 gga gga gtg gta gtg agc gct gtg ttc ttc att ttg tcggcc aac att 579 Gly Gly Val Val Val Ser Ala Val Phe Phe Ile Leu Ser AlaAsn Ile 175 180 185 190 cta tca tct ccc tct aag aga gga cag aaa ggt accctt att gga tat 627 Leu Ser Ser Pro Ser Lys Arg Gly Gln Lys Gly Thr LeuIle Gly Tyr 195 200 205 tct cct gaa gga aca cca ctc tat cac ttc atg ggggac gct ttt cag 675 Ser Pro Glu Gly Thr Pro Leu Tyr His Phe Met Gly AspAla Phe Gln 210 215 220 cac agc tct cag tcg gtg cct agg ttc att aag gactca cta aag cag 723 His Ser Ser Gln Ser Val Pro Arg Phe Ile Lys Asp SerLeu Lys Gln 225 230 235 gtt ctc gag gag agc gac tct agg cag atc ttt tacttc ttg tgc ttg 771 Val Leu Glu Glu Ser Asp Ser Arg Gln Ile Phe Tyr PheLeu Cys Leu 240 245 250 aat ctg ctt ttt acc ttt gtg gag ttg ttc tat ggcgtg cta acc aac 819 Asn Leu Leu Phe Thr Phe Val Glu Leu Phe Tyr Gly ValLeu Thr Asn 255 260 265 270 agt cta ggc ctg atc tca gat gga ttt cac atgctc ttt gac tgc tcg 867 Ser Leu Gly Leu Ile Ser Asp Gly Phe His Met LeuPhe Asp Cys Ser 275 280 285 gct ttg gtc atg gga ctg ttt gct gcc ctg atgagc cgc tgg aaa gcc 915 Ala Leu Val Met Gly Leu Phe Ala Ala Leu Met SerArg Trp Lys Ala 290 295 300 acc cgg att ttc tcc tat ggg tat ggc cga atagag att ctc tct ggc 963 Thr Arg Ile Phe Ser Tyr Gly Tyr Gly Arg Ile GluIle Leu Ser Gly 305 310 315 ttt att aat ggg ctt ttt ctg atc gtg ata gcattt ttt gtg ttt atg 1011 Phe Ile Asn Gly Leu Phe Leu Ile Val Ile Ala PhePhe Val Phe Met 320 325 330 gaa tca gtg gct aga ctg atc gat cct ccg gaacta gac aca aac atg 1059 Glu Ser Val Ala Arg Leu Ile Asp Pro Pro Glu LeuAsp Thr Asn Met 335 340 345 350 ctg aca cca gtt tcc gtc gca ggg ctg atagta aac ctt att ggt atc 1107 Leu Thr Pro Val Ser Val Ala Gly Leu Ile ValAsn Leu Ile Gly Ile 355 360 365 tgt gcc ttc agc cac gcc cac agc cat ggccat ggc gct tct caa gga 1155 Cys Ala Phe Ser His Ala His Ser His Gly HisGly Ala Ser Gln Gly 370 375 380 aac tgc cac tct gat cac ggc cat tca caccat gca cat gga cac ggc 1203 Asn Cys His Ser Asp His Gly His Ser His HisAla His Gly His Gly 385 390 395 cat gat cac ggt cac agc cac ggc ttc acgggt gga ggc atg aat gcg 1251 His Asp His Gly His Ser His Gly Phe Thr GlyGly Gly Met Asn Ala 400 405 410 aac atg agg ggt gta ttt ctc cat gtg ttggca gac aca ctg ggc agc 1299 Asn Met Arg Gly Val Phe Leu His Val Leu AlaAsp Thr Leu Gly Ser 415 420 425 430 atc ggc gtg att gtg tcc aca gtt ctcata gag cag ttt gga tgg ttc 1347 Ile Gly Val Ile Val Ser Thr Val Leu IleGlu Gln Phe Gly Trp Phe 435 440 445 att gct gat ccc ctg tgt tct ctt tttatt gcc gtg ttg ata ttt ctc 1395 Ile Ala Asp Pro Leu Cys Ser Leu Phe IleAla Val Leu Ile Phe Leu 450 455 460 agt gtg atc cca ctg att aaa gat gcctgt caa gtt cta ctt ctg aga 1443 Ser Val Ile Pro Leu Ile Lys Asp Ala CysGln Val Leu Leu Leu Arg 465 470 475 cta cca cct gac cat gaa aaa gaa ctgcat att gct tta gaa aag ata 1491 Leu Pro Pro Asp His Glu Lys Glu Leu HisIle Ala Leu Glu Lys Ile 480 485 490 cag aaa att gag gga tta ata tca taccga gac cct cat ttt tgg cgc 1539 Gln Lys Ile Glu Gly Leu Ile Ser Tyr ArgAsp Pro His Phe Trp Arg 495 500 505 510 cat tct gcc agt att gta gcg ggaaca att cat ata caa gtg aca tct 1587 His Ser Ala Ser Ile Val Ala Gly ThrIle His Ile Gln Val Thr Ser 515 520 525 gag gtg ctg gag cag aga att gtacag cag gtt aca ggg ata ctt aaa 1635 Glu Val Leu Glu Gln Arg Ile Val GlnGln Val Thr Gly Ile Leu Lys 530 535 540 gat gca gga gta aac aac cta accata caa gtg gaa aag gag gca tac 1683 Asp Ala Gly Val Asn Asn Leu Thr IleGln Val Glu Lys Glu Ala Tyr 545 550 555 ttt cag cat atg tcc ggc ct 1703Phe Gln His Met Ser Gly 560 <210> SEQ ID NO 28 <211> LENGTH: 564 <212>TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 28 Met Ala LysMet Ala Glu His Pro Glu Gly His His Asp Ser Ala Leu 1 5 10 15 Thr HisMet Leu Tyr Thr Ala Ile Ala Phe Leu Gly Val Ala Asp His 20 25 30 Lys GlyGly Val Leu Leu Leu Val Leu Ala Leu Cys Cys Lys Val Gly 35 40 45 Phe HisThr Ala Ser Arg Lys Leu Ser Ile Asp Val Gly Gly Ala Lys 50 55 60 Arg LeuGln Ala Leu Ser Gln Leu Val Ser Val Phe Leu Leu Cys Pro 65 70 75 80 TrpVal Ile Val Leu Ser Val Thr Thr Glu Ser Lys Val Glu Ser Trp 85 90 95 PheSer Leu Ile Met Pro Phe Thr Thr Val Ile Phe Phe Val Met Ile 100 105 110Leu Asp Phe Tyr Met Asp Ser Val Cys Ser Val Lys Met Asp Val Ser 115 120125 Lys Cys Ala Arg Tyr Gly Ser Phe Pro Ile Phe Ile Ser Ala Leu Leu 130135 140 Phe Arg Asn Phe Trp Thr His Pro Ile Thr Asp Gln Leu Arg Ala Met145 150 155 160 Asn Arg Ala Ala His Gln Glu Ser Thr Glu His Val Leu SerGly Gly 165 170 175 Val Val Val Ser Ala Val Phe Phe Ile Leu Ser Ala AsnIle Leu Ser 180 185 190 Ser Pro Ser Lys Arg Gly Gln Lys Gly Thr Leu IleGly Tyr Ser Pro 195 200 205 Glu Gly Thr Pro Leu Tyr His Phe Met Gly AspAla Phe Gln His Ser 210 215 220 Ser Gln Ser Val Pro Arg Phe Ile Lys AspSer Leu Lys Gln Val Leu 225 230 235 240 Glu Glu Ser Asp Ser Arg Gln IlePhe Tyr Phe Leu Cys Leu Asn Leu 245 250 255 Leu Phe Thr Phe Val Glu LeuPhe Tyr Gly Val Leu Thr Asn Ser Leu 260 265 270 Gly Leu Ile Ser Asp GlyPhe His Met Leu Phe Asp Cys Ser Ala Leu 275 280 285 Val Met Gly Leu PheAla Ala Leu Met Ser Arg Trp Lys Ala Thr Arg 290 295 300 Ile Phe Ser TyrGly Tyr Gly Arg Ile Glu Ile Leu Ser Gly Phe Ile 305 310 315 320 Asn GlyLeu Phe Leu Ile Val Ile Ala Phe Phe Val Phe Met Glu Ser 325 330 335 ValAla Arg Leu Ile Asp Pro Pro Glu Leu Asp Thr Asn Met Leu Thr 340 345 350Pro Val Ser Val Ala Gly Leu Ile Val Asn Leu Ile Gly Ile Cys Ala 355 360365 Phe Ser His Ala His Ser His Gly His Gly Ala Ser Gln Gly Asn Cys 370375 380 His Ser Asp His Gly His Ser His His Ala His Gly His Gly His Asp385 390 395 400 His Gly His Ser His Gly Phe Thr Gly Gly Gly Met Asn AlaAsn Met 405 410 415 Arg Gly Val Phe Leu His Val Leu Ala Asp Thr Leu GlySer Ile Gly 420 425 430 Val Ile Val Ser Thr Val Leu Ile Glu Gln Phe GlyTrp Phe Ile Ala 435 440 445 Asp Pro Leu Cys Ser Leu Phe Ile Ala Val LeuIle Phe Leu Ser Val 450 455 460 Ile Pro Leu Ile Lys Asp Ala Cys Gln ValLeu Leu Leu Arg Leu Pro 465 470 475 480 Pro Asp His Glu Lys Glu Leu HisIle Ala Leu Glu Lys Ile Gln Lys 485 490 495 Ile Glu Gly Leu Ile Ser TyrArg Asp Pro His Phe Trp Arg His Ser 500 505 510 Ala Ser Ile Val Ala GlyThr Ile His Ile Gln Val Thr Ser Glu Val 515 520 525 Leu Glu Gln Arg IleVal Gln Gln Val Thr Gly Ile Leu Lys Asp Ala 530 535 540 Gly Val Asn AsnLeu Thr Ile Gln Val Glu Lys Glu Ala Tyr Phe Gln 545 550 555 560 His MetSer Gly <210> SEQ ID NO 29 <211> LENGTH: 29 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence:an artificially synthesized primersequence <220> FEATURE: <221> NAME/KEY: primer_bind <222> LOCATION:(1)..(29) <400> SEQUENCE: 29 gggaattcat gggtaagacc ctgcggggc 29 <210>SEQ ID NO 30 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence:an artificially synthesized primer sequence <220>FEATURE: <221> NAME/KEY: primer_bind <222> LOCATION: (1)..(35) <400>SEQUENCE: 30 ggaagctttc actcatgtaa cccagattct ccagc 35 <210> SEQ ID NO31 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence:an artificially synthesized primer sequence <220> FEATURE:<221> NAME/KEY: primer_bind <222> LOCATION: (1)..(35) <400> SEQUENCE: 31ggttttctag agctcgtgca gacctggaga cgggc 35 <210> SEQ ID NO 32 <211>LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:anartificially synthesized primer sequence <220> FEATURE: <221> NAME/KEY:primer_bind <222> LOCATION: (1)..(39) <400> SEQUENCE: 32 gggttttctagatcttcaga aatgagggcg acttttgtg 39 <210> SEQ ID NO 33 <211> LENGTH: 29<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:an artificiallysynthesized primer sequence <220> FEATURE: <221> NAME/KEY: primer_bind<222> LOCATION: (1)..(29) <400> SEQUENCE: 33 gggatccgca tagtacagcaggttacagg 29 <210> SEQ ID NO 34 <211> LENGTH: 29 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence:an artificially synthesized primersequence <220> FEATURE: <221> NAME/KEY: primer_bind <222> LOCATION:(1)..(29) <400> SEQUENCE: 34 ggtcgaccct ttaaaataca taactcaaa 29 <210>SEQ ID NO 35 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence:an artificially synthesized primer sequence <220>FEATURE: <221> NAME/KEY: primer_bind <222> LOCATION: (1)..(30) <400>SEQUENCE: 35 aatgtatgca gtatggaaat ggaaagttgc 30 <210> SEQ ID NO 36<211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence:an artificially synthesized primer sequence <220> FEATURE:<221> NAME/KEY: primer_bind <222> LOCATION: (1)..(28) <400> SEQUENCE: 36cgaatgggct gaccgcttcc tcgtgctt 28 <210> SEQ ID NO 37 <211> LENGTH: 30<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:an artificiallysynthesized primer sequence <220> FEATURE: <221> NAME/KEY: primer_bind<222> LOCATION: (1)..(30) <400> SEQUENCE: 37 cggcaggagc aaggtgagatgacaggagat 30 <210> SEQ ID NO 38 <211> LENGTH: 30 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence:an artificially synthesized primersequence <220> FEATURE: <221> NAME/KEY: primer_bind <222> LOCATION:(1)..(30) <400> SEQUENCE: 38 ttacagtgtc aggaataaag gctatgcttc 30 <210>SEQ ID NO 39 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence:an artificially synthesized primer sequence <220>FEATURE: <221> NAME/KEY: primer_bind <222> LOCATION: (1)..(20) <400>SEQUENCE: 39 gactcgatct catggcaaag 20 <210> SEQ ID NO 40 <211> LENGTH:21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence:anartificially synthesized primer sequence <220> FEATURE: <221> NAME/KEY:primer_bind <222> LOCATION: (1)..(21) <400> SEQUENCE: 40 tagagcatgtgagttagagc a 21 <210> SEQ ID NO 41 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:an artificiallysynthesized primer sequence <220> FEATURE: <221> NAME/KEY: primer_bind<222> LOCATION: (1)..(21) <400> SEQUENCE: 41 ccaacattct atcatctccc t 21<210> SEQ ID NO 42 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence:an artificially synthesized primer sequence <220>FEATURE: <221> NAME/KEY: primer_bind <222> LOCATION: (1)..(19) <400>SEQUENCE: 42 agcggctcat cagggcagc 19

1. A DNA encoding a protein having the amino acid sequence of SEQ ID NO:4.
 2. A DNA encoding a protein fragment comprising the extracellularregion of a protein having the amino acid sequence of SEQ ID NO:
 4. 3. ADNA comprising a nucleotide sequence corresponding to nucleotideresidues 97 to 1419 of the nucleotide sequence of SEQ ID NO:
 3. 4. A DNAhybridizing with a DNA having the nucleotide sequence of SEQ ID NO: 3under stringent conditions.
 5. A protein having the amino acid sequenceof SEQ ID NO: 4 or an amino acid sequence substantially the same as saidamino acid sequence.
 6. A protein fragment comprising the extracellularregion of a protein having the amino acid sequence of SEQ ID NO: 4 or anamino acid sequence substantially the same as said amino acid sequence.7. A fusion protein between the extracellular region of the protein ofclaim 5 and the constant region of the heavy chain of humanimmunoglobulin (Ig) or a portion of the constant region.
 8. Anexpression vector comprising the DNA of any one of claims 1 to
 4. 9. Atransformant carrying the expression vector of claim
 8. 10. An antibodyor its portion reactive with the protein of claim 5 or the proteinfragment of claim
 6. 11. The antibody or its portion of claim 10,wherein the antibody is a monoclonal antibody.
 12. A pharmaceuticalcomposition comprising the protein fragment of claim 6 or the fusionprotein of claim 7 and a pharmaceutically acceptable carrier.
 13. Apharmaceutical composition comprising the antibody or its portion ofclaim 10 or 11 and a pharmaceutically acceptable carrier.
 14. A DNAencoding a protein having the amino acid sequence of SEQ ID NO:
 10. 15.A DNA encoding a protein fragment comprising the extracellular region ofa protein having the amino acid sequence of SEQ ID NO:
 10. 16. A DNAhaving a nucleotide sequence corresponding to nucleotide residues 1 to1785 of the nucleotide sequence of SEQ ID NO:
 9. 17. A DNA hybridizingwith a DNA having the nucleotide sequence of SEQ ID NO: 9 understringent conditions.
 18. A protein having the amino acid sequence ofSEQ ID NO: 10 or an amino acid sequence substantially the same as saidamino acid sequence.
 19. A protein fragment comprising the extracellularregion of a protein having the amino acid sequence of SEQ ID NO: 10 oran amino acid sequence substantially the same as said amino acidsequence.
 20. A fusion protein comprising the extracellular region ofthe protein of claim 18 and the constant region of the heavy chain ofhuman immunoglobulin (Ig) or a portion of the constant region.
 21. Anexpression vector comprising the DNA of any one of claims 14 to
 17. 22.A transformant carrying the expression vector of claim
 21. 23. Anantibody or its portion reactive with the protein of claim 18 or theprotein fragment of claim
 19. 24. The antibody or its portion of claim23, wherein the antibody is a monoclonal antibody.
 25. A pharmaceuticalcomposition comprising the protein fragment of claim 19 or the fusionprotein of claim 20 and a pharmaceutically acceptable carrier.
 26. Apharmaceutical composition comprising the antibody or its portion ofclaim 23 or 24 and a pharmaceutically acceptable carrier.
 27. Atransgenic mouse in which the human-derived DNA comprising a DNA havinga nucleotide sequence corresponding to nucleotide residues 97 to 1419 ofthe nucleotide sequence of SEQ ID NO: 3 is integrated into an endogenousgene of said mouse.
 28. A transgenic mouse in which the human-derivedDNA comprising a DNA having a nucleotide sequence corresponding tonucleotide residues 1 to 1785 of the nucleotide sequence of SEQ ID NO: 9is integrated into an endogenous gene of said mouse.
 29. A knockoutmouse whose endogenous gene encoding a mouse-derived protein having theamino acid sequence of SEQ ID NO: 6 is inactivated so that said proteinis not produced.
 30. A knockout mouse whose endogenous gene encoding amouse-derived protein comprising the amino acid sequence of SEQ ID NO:28 is inactivated so that said protein is not produced.